Production of carotenoids in oleaginous yeast and fungi

ABSTRACT

The present invention provides systems for producing engineered oleaginous yeast or fungi that express carotenoids

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/663,621, filed Mar. 18, 2005, the contents of which are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Carotenoids are organic pigments ranging in color from yellow to redthat are naturally produced by certain organisms, includingphotosynthetic organisms (e.g., plants, algae, cyanobacteria), and somefungi. Carotenoids are responsible for the orange color of carrots, aswell as the pink in flamingos and salmon, and the red in lobsters andshrimp. Animals, however, cannot produce carotenoids and must receivethem through their diet.

Carotenoid pigments (e.g., β-carotene and astaxanthin) are usedindustrially as ingredients for food and feed stocks, both serving anutritional function and enhancing consumer acceptability. For example,astaxanthin is widely used in salmon aquaculture to provide the orangecoloration characteristic of their wild counterparts. Some carotenoidsare also precursors of vitamin A. Also, carotenoids have antioxidantproperties, and may have various health benefits (see, for example,Jyonouchi et al., Nutr. Cancer 16:93, 1991; Giovannucci et al., J. Natl.Cancer Inst. 87:1767, 1995; Miki, Pure Appl. Chem. 63:141, 1991; Chew etal., Anticancer Res. 19:1849, 1999; Wang et al., Antimicrob. AgentsChemother. 44:2452, 2000). Some carotenoids such as β-carotene,lycopene, and lutein are currently sold as nutritional supplements.

In general, the biological systems that produce carotenoids areindustrially intractable and/or produce the compounds at such low levelsthat commercial scale isolation is not practicable. Thus, mostcarotenoids used in industry are produced by chemical synthesis. Thereis a need for improved biological systems that produce carotenoids. Someefforts have previously been made to genetically engineer certainbacteria or fungi to produce higher levels of carotenoids (see, forexample, Misawa et al., J. Biotechnol. 59:169, 1998; Visser et al., FEMSYeast Research 4:221, 2003). However, improved systems, allowing higherlevels of production and greater ease of isolation, are needed.

SUMMARY OF THE INVENTION

The present invention provides improved systems for the biologicalproduction of carotenoids. In one aspect, the invention encompasses thediscovery that it is desirable to produce carotenoids in oleaginousorganisms. Without wishing to be bound by any particular theory, thepresent inventors propose that biological systems may be able toaccumulate higher levels of carotenoids if the compounds are sequesteredin lipid bodies. Regardless of whether absolute levels are higher,however, carotenoids that are accumulated within lipid bodies inoleaginous organisms are readily isolatable through isolation of thelipid bodies.

The present invention therefore provides oleaginous fungi (including,for example, yeast or other unicellular fungi) that produce one or morecarotenoids. The present invention also provides methods of constructingsuch yeast and fungi, methods of using such yeast and fungi to producecarotenoids, and methods of preparing carotenoid-containingcompositions, such as food or feed additives, or nutritionalsupplements, using carotenoids produced in such oleaginous yeast orfungi. In particular, the present invention provides systems and methodsfor generating yeast and fungi containing one or more oleaginic and/orcarotenogenic modifications that increase the oleaginicity and/or altertheir carotenoid-producing capabilities as compared with otherwiseidentical organisms that lack the modification(s).

The present invention further encompasses the general recognition thatlipid-accumulating systems are useful for the production and/orisolation of lipophilic agents (such as, but not limited to isoprenoids,or isoprenoid-derived compounds). Thus, according to the presentinvention, it is desirable to engineer organisms to produce suchlipophilic agents and/or to accumulate lipid.

Various other aspects of the present invention will be apparent to thoseof ordinary skill in the art from the present description, including theappended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A-1D depicts certain common carotenoids.

FIG. 2 depicts how sufficient levels of acetyl-CoA and NADPH may beaccumulated in the cytosol of oleaginous organisms to allow forproduction of significant levels of cytosolic lipids. Enzymes: 1,pyruvate decarboxylase; 2, malate dehydrogenase; 3, malic enzyme; 4,pyruvate dehydrogenase; 5, citrate synthase; 6, ATP-citrate lyase; 7,citrate/malate translocase.

FIGS. 3A and 3B depict the mevalonate isoprenoid biosynthesis pathway,which typically operates in eukaryotes, including fungi.

FIG. 4 depicts the mevalonate-independent isoprenoid biosynthesispathway, also known as the DXP pathway, which typically operates inbacteria and in the plastids of plants.

FIG. 5 depicts intermediates in the isoprenoid biosynthesis pathway andhow they feed into biosynthetic pathways of other biomolecules,including carotenoids as well as non-carotenoid compounds such assterols, steroids, and vitamins, such as vitamin E or vitamin K.

FIGS. 6A-6D illustrate various carotenoid biosynthetic pathways. FIG. 6Ahighlights branches leading to various cyclic and acyclic xanthophylls;FIG. 6B shows certain X. dendrorhous pathways that generate dicyclic andmonocyclic carotenoids, including astaxanthin; FIG. 6C showsinterconnecting pathways for converting β-carotene into any of a varietyof other carotenoids, including astaxanthin; FIG. 6D depicts possibleroutes of synthesis of cyclic carotenoids and common plant and algalxanthophylls from neurosporene.

FIGS. 7A-7C show an alignment of certain representative fungal HMG-CoAreductase polypeptides. As can be seen, these polypeptides show veryhigh identity across the catalytic region, and also have complexmembrane spanning domains. In some embodiments of the invention, thesemembrane-spanning domains are disrupted or are removed, so that, forexample, a hyperactive version of the polypeptide may be produced.

FIGS. 8A-8D depict schematic representations of plasmids generated anddescribed in detail in the exemplification.

DEFINITIONS

Carotenogenic modification: The term “carotenogenic modification”, asused herein, refers to a modification of a host organism that adjustsproduction of one or more carotenoids, as described herein. For example,a carotenogenic modification may increase the production level of one ormore carotenoids, and/or may alter relative production levels ofdifferent carotenoids. In principle, an inventive carotenogenicmodification may be any chemical, physiological, genetic, or othermodification that appropriately alters production of one or morecarotenoids in a host organism produced by that organism as comparedwith the level produced in an otherwise identical organism not subjectto the same modification. In most embodiments, however, thecarotenogenic modification will comprise a genetic modification,typically resulting in increased production of one or more selectedcarotenoids. In some embodiments, the selected carotenoid is one or moreof astaxanthin, β-carotene, canthaxanthin, lutein, lycopene, phytoene,zeaxanthin, and/or modifications of zeaxanthin or astaxanthin (e.g.,glucoside, esterified zeaxanthin or astaxanthin). In some embodiments,the selected carotenoid is one or more xanthophylls, and/or amodification thereof (e.g., glucoside, esterified xanthophylls). Incertain embodiments, the selected xanthophyll is selected from the groupconsisting of astaxanthin, lutein, zeaxanthin, lycopene, andmodifications thereof. In some embodiments, the selected carotenoid isone or more of astaxanthin, β-carotene, canthaxanthin, lutein, lycopene,and zeaxanthin and/or modifications of zeaxanthin or astaxanthin. Insome embodiments, the carotenoid is β-carotene. In some embodiments, theselected carotenoid is astaxanthin. In some embodiments, the selectedcarotenoid is other than β-carotene.

Carotenogenic polypeptide: The term “carotenogenic polypeptide”, as usedherein, refers to any polypeptide that is involved in the process ofproducing carotenoids in a cell, and may include polypeptides that areinvolved in processes other than carotenoid production but whoseactivities affect the extent or level of production of one or morecarotenoids, for example by scavenging a substrate or reactant utilizedby a carotenoid polypeptide that is directly involved in carotenoidproduction. Carotenogenic polypeptides include isoprenoid biosynthesispolypeptides, carotenoid biosynthesis polypeptides, and isoprenoidbiosynthesis competitor polypeptides, as those terms are defined herein.The term also encompasses polypeptides that may affect the extent towhich carotenoids are accumulated in lipid bodies.

Carotenoid: The term “carotenoid” is understood in the art to refer to astructurally diverse class of pigments derived from isoprenoid pathwayintermediates. The commitment step in carotenoid biosynthesis is theformation of phytoene from geranylgeranyl pyrophosphate. Carotenoids canbe acyclic or cyclic, and may or may not contain oxygen, so that theterm carotenoids include both carotenes and xanthophylls. In general,carotenoids are hydrocarbon compounds having a conjugated polyene carbonskeleton formally derived from the five-carbon compound IPP, includingtriterpenes (C₃₀ diapocarotenoids) and tetraterpenes (C₄₀ carotenoids)as well as their oxygenated derivatives and other compounds that are,for example, C₃₅, C₅₀, C₆₀, C₇₀, C₈₀ in length or other lengths. Manycarotenoids have strong light absorbing properties and may range inlength in excess of C₂₀₀. C₃₀ diapocarotenoids typically consist of sixisoprenoid units joined in such a manner that the arrangement ofisoprenoid units is reversed at the center of the molecule so that thetwo central methyl groups are in a 1,6-positional relationship and theremaining non-terminal methyl groups are in a 1,5-positionalrelationship. Such C₃₀ carotenoids may be formally derived from theacyclic C₃₀H₄₂ structure, having a long central chain of conjugateddouble bonds, by: (i) hydrogenation (ii) dehydrogenation, (iii)cyclization, (iv) oxidation, (v) esterification/glycosylation, or anycombination of these processes. C₄₀ carotenoids typically consist ofeight isoprenoid units joined in such a manner that the arrangement ofisoprenoid units is reversed at the center of the molecule so that thetwo central methyl groups are in a 1,6-positional relationship and theremaining non-terminal methyl groups are in a 1,5-positionalrelationship. Such C₄₀ carotenoids may be formally derived from theacyclic C₄₀H₅₆ structure, having a long central chain of conjugateddouble bonds, by (i) hydrogenation, (ii) dehydrogenation, (iii)cyclization, (iv) oxidation, (v) esterification/glycosylation, or anycombination of these processes. The class of C₄₀ carotenoids alsoincludes certain compounds that arise from rearrangements of the carbonskeleton, or by the (formal) removal of part of this structure. Morethan 600 different carotenoids have been identified in nature; certaincommon carotenoids are depicted in FIG. 1. Carotenoids include but arenot limited to: antheraxanthin, adonirubin, adonixanthin, astaxanthin,canthaxanthin, capsorubrin, β-cryptoxanthin, α-carotene, β-carotene,β,ψ-carotene, δ-carotene, ε-carotene, echinenone, 3-hydroxyechinenone,3′-hydroxyechinenone, γ-carotene, ψ-carotene, 4-keto-γ-carotene,ζ-carotene, α-cryptoxanthin, deoxyflexixanthin, diatoxanthin,7,8-didehydroastaxanthin, didehydrolycopene, fucoxanthin, fucoxanthinol,isorenieratene, β-isorenieratene, lactucaxanthin, lutein, lycopene,myxobactone, neoxanthin, neurosporene, hydroxyneurosporene, peridinin,phytoene, rhodopin, rhodopin glucoside, 4-keto-rubixanthin,siphonaxanthin, spheroidene, spheroidenone, spirilloxanthin, torulene,4-keto-torulene, 3-hydroxy-4-keto-torulene, uriolide, uriolide acetate,violaxanthin, zeaxanthin-β-diglucoside, zeaxanthin, and C30 carotenoids.Additionally, carotenoid compounds include derivatives of thesemolecules, which may include hydroxy-, methoxy-, oxo-, epoxy-, carboxy-,or aldehydic functional groups. Further, included carotenoid compoundsinclude ester (e.g., glycoside ester, fatty acid ester) and sulfatederivatives (e.g., esterified xanthophylls).

Carotenoid biosynthesis polypeptide: The term “carotenoid biosynthesispolypeptide” refers to any polypeptide that is involved in the synthesisof one or more carotenoids. To mention but a few, these carotenoidbiosynthesis polypeptides include, for example, polypeptides of phytoenesynthase, phytoene dehydrogenase (or desaturase), lycopene cyclase,carotenoid ketolase, carotenoid hydroxylase, astaxanthin synthase,carotenoid epsilon hydroxylase, lycopene cyclase (beta and epsilonsubunits), carotenoid glucosyltransferase, and acyl CoA:diacyglycerolacyltransferase. Representative examples of carotenoid biosynthesispolypeptide sequences are presented in Tables 17-25.

Gene: The term “gene”, as used herein, generally refers to a nucleicacid encoding a polypeptide, optionally including certain regulatoryelements that may affect expression of one or more gene products (i.e.,RNA or protein).

Heterologous: The term “heterologous”, as used herein to refer to genesor polypeptides, refers to a gene or polypeptide that does not naturallyoccur in the organism in which it is being expressed. It will beunderstood that, in general, when a heterologous gene or polypeptide isselected for introduction into and/or expression by a host cell, theparticular source organism from which the heterologous gene orpolypeptide may be selected is not essential to the practice of thepresent invention. Relevant considerations may include, for example, howclosely related the potential source and host organisms are inevolution, or how related the source organism is with other sourceorganisms from which sequences of other relevant polypeptides have beenselected.

Host cell: As used herein, the “host cell” is a yeast or fungal cellthat is manipulated according to the present invention to accumulatelipid and/or to express one or more carotenoids as described herein. A“modified host cell”, as that term is used herein, is a host cell thatcontains at least one oleaginic modification and/or at least onecarotenogenic modification according to the present invention.

Isolated: The term “isolated”, as used herein, means that the isolatedentity has been separated from at least one component with which it waspreviously associated. When most other components have been removed, theisolated entity is “purified”. Isolation and/or purification may beperformed using any techniques known in the art including, for example,fractionation, extraction, precipitation, or other separation.

Isoprenoid biosynthesis competitor polypeptide: The term “isoprenoidbiosynthesis competitor polypeptide”, as used herein, refers to apolypeptide whose expression in a cell reduces the level ofgeranylgeranyl diphosphate (GGPP) available to enter the carotenoidbiosynthesis pathway. For example, isoprenoid biosynthesis competitorpolypeptides include enzymes that act on isoprenoid intermediates priorto GGPP, such that less GGPP is generated (see, for example, FIG. 5).Squalene synthase is but one isoprenoid biosynthesis competitorpolypeptide according to the present invention; representative squalenesynthase sequences are presented in Table 16. Prenyldiphosphate synthaseenzymes and para-hydroxybenzoate (PHB) polyprenyltransferase are yetadditional isoprenoid biosynthesis competitor polypeptides according tothe present invention; representative prenyldiphosphate synthase enzymesand PHB polyprenyltransferase polypeptides are presented in Table 29 and30 respectively.

Isoprenoid biosynthesis polypeptide: The term “isoprenoid biosynthesispolypeptide” refers to any polypeptide that is involved in the synthesisof isoprenoids. For example, as discussed herein, acetoacetyl-CoAthiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase,phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, IPPisomerase, FPP synthase, and GGPP synthase, are all involved in themevalonate pathway for isoprenoid biosynthesis. Each of these proteinsis also an isoprenoid biosynthesis polypeptide for purposes of thepresent invention, and sequences of representative examples of theseenzymes are provided in Tables 7-15.

Isoprenoid pathway: The “isoprenoid pathway” is understood in the art torefer to a metabolic pathway that either produces or utilizes thefive-carbon metabolite isopentyl pyrophosphate (IPP). As discussedherein, two different pathways can produce the common isoprenoidprecursor IPP—the “mevalonate pathway” and the “non-mevalonate pathway”.The term “isoprenoid pathway” is sufficiently general to encompass bothof these types of pathway. Biosynthesis of isoprenoids from IPP occursby polymerization of several five-carbon isoprene subunits. Isoprenoidmetabolites derived from IPP are of varying size and chemical structure,including both cyclic and acyclic molecules. Isoprenoid metabolitesinclude, but are not limited to, monoterpenes, sesquiterpenes,diterpenes, sterols, and polyprenols such as carotenoids.

Oleaginic modification: The term “oleaginic modification”, as usedherein, refers to a modification of a host organism that adjusts thedesirable oleaginy of that host organism, as described herein. In somecases, the host organism will already be oleaginous in that it will havethe ability to accumulate lipid to at least about 20% of its dry cellweight. It may nonetheless be desirable to apply an oleaginicmodification to such an organism, in accordance with the presentinvention, for example to increase (or, in some cases, possibly todecrease) its total lipid accumulation, or to adjust the types oramounts of one or more particular lipids it accumulates (e.g., toincrease relative accumulation of triacylglycerol). In other cases, thehost organism may be non-oleaginous (though may contain some enzymaticand regulatory components used in other organisms to accumulate lipid),and may require oleaginic modification in order to become oleaginous inaccordance with the present invention. The present invention alsocontemplates application of oleaginic modification to non-oleaginoushost strains such that their oleaginicity is increased even though, evenafter being modified, they may not be oleaginous as defined herein. Inprinciple, the oleaginic modification may be any chemical,physiological, genetic, or other modification that appropriately altersoleaginy of a host organism as compared with an otherwise identicalorganism not subjected to the oleaginic modification. In mostembodiments, however, the oleaginic modification will comprise a geneticmodification, typically resulting in increased production and/oractivity of one or more oleaginic polypeptides. In some embodiments, theoleaginic modification comprises at least one chemical, physiological,genetic, or other modification; in other embodiments, the oleaginicmodification comprises more than one chemical, physiological, genetic,or other modification. In certain aspects where more than onemodification is utilized, such modifications can comprise anycombination of chemical, physiological, genetic, or other modification(e.g., one or more genetic modification and chemical or physiologicalmodification).

Oleaginic polypeptide: The term “oleaginic polypeptide”, as used herein,refers to any polypeptide that is involved in the process of lipidaccumulation in a cell and may include polypeptides that are involved inprocesses other than lipid biosynthesis but whose activities affect theextent or level of accumulation of one or more lipids, for example byscavenging a substrate or reactant utilized by an oleaginic polypeptidethat is directly involved in lipid accumulation. For example, asdiscussed herein, acetyl-CoA carboxylase, pyruvate decarboxylase,isocitrate dehydrogenase, ATP-citrate lyase, malic enzyme, and AMPdeaminase, among other proteins, are all involved in lipid accumulationin cells. In general, reducing the activity of pyruvate decarboxylase orisocitrate dehydrogenase, and/or increasing the activity of acetyl CoAcarboxylase, ATP-citrate lyase, malic enzyme and/or AMP deaminase isexpected to promote oleaginy. Each of these proteins is an oleaginicpolypeptide for purposes of the present invention, and sequences ofrepresentative examples of these enzymes are provided in Tables 1-6.

Oleaginous: The term “oleaginous”, refers to the ability of an organismto accumulate lipid to at least about 20% of its dry cell weight. Incertain embodiments of the invention, oleaginous yeast or fungiaccumulate lipid to at least about 25% of their dry cell weight. Inother embodiments, inventive oleaginous yeast or fungi accumulate lipidwithin the range of about 20-45% of their dry cell weight. In someembodiments, oleaginous organisms may accumulate lipid to as much asabout 70% of their dry cell weight. In some embodiments of theinvention, oleaginous organisms may accumulate a large fraction of totallipid accumulation in the form of triacylglycerol. In certainembodiments, the majority of the accumulated lipid is in the form oftriacylglycerol. Alternatively or additionally, the lipid may accumulatein the form of intracellular lipid bodies, or oil bodies. In certainembodiments, the present invention utilizes yeast or fungi that arenaturally oleaginous. In some aspects, naturally oleaginous organismsare manipulated (e.g., genetically, chemically, or otherwise) so as tofurther increase the level of accumulated lipid in the organism. Inother embodiments, yeast or fungi that are not naturally oleaginous aremanipulated (e.g., genetically, chemically, or otherwise) to accumulatelipid as described herein. For the purposes of the present invention,Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and Candida utilis arenot naturally oleaginous fungi.

Polypeptide: The term “polypeptide”, as used herein, generally has itsart-recognized meaning of a polymer of at least three amino acids.However, the term is also used to refer to specific functional classesof polypeptides, such as, for example, oleaginic polypeptides,carotenogenic polypeptides, isoprenoid biosynthesis polypeptides,carotenoid biosynthesis polypeptides, and isoprenoid biosynthesiscompetitor polypeptides. For each such class, the present specificationprovides several examples of known sequences of such polypeptides. Thoseof ordinary skill in the art will appreciate, however, that the term“polypeptide” is intended to be sufficiently general as to encompass notonly polypeptides having the complete sequence recited herein (or in areference or database specifically mentioned herein), but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those of ordinary skill in the art understandthat protein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90% or even 95%,96%, 97%, 98%, or 99% in one or more highly conserved regions (e.g.,isocitrate dehydrogenase polypeptides often share a conservedAMP-binding motif; HMG-CoA reductase polypeptides typically include ahighly conserved catalytic domain (see, for example, FIG. 7); acetyl coAcarboxylase typically has a carboxyl transferase domain; see, forexample, Downing et al., Chem. Abs. 93:484, 1980; Gil et al., Cell41:249, 1985; Jitrapakdee et al. Curr Protein Pept Sci. 4:217, 2003;U.S. Pat. No. 5,349,126, each of which is incorporated herein byreference in its entirety), usually encompassing at least 3-4 and oftenup to 20 or more amino acids, with another polypeptide of the sameclass, is encompassed within the relevant term “polypeptide” as usedherein.

Source organism: The term “source organism”, as used herein, refers tothe organism in which a particular polypeptide sequence can be found innature. Thus, for example, if one or more heterologous polypeptidesis/are being expressed in a host organism, the organism in which thepolypeptides are expressed in nature (and/or from which their genes wereoriginally cloned) is referred to as the “source organism”. Where morethan one heterologous polypeptides are being expressed in a hostorganism, one or more source organism(s) may be utilized for independentselection of each of the heterologous polypeptide(s). It will beappreciated that any and all organisms that naturally contain relevantpolypeptide sequences may be used as source organisms in accordance withthe present invention. Representative source organisms include, forexample, animal, mammalian, insect, plant, fungal, yeast, algal,bacterial, cyanobacterial, archaebacterial and protozoal sourceorganisms.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

As noted above, the present invention encompasses the discovery thatcarotenoids can desirably be produced in oleaginous yeast and fungi.According to the present invention, strains that both (i) accumulatelipid, often in the form of cytoplasmic oil bodies and typically to atleast about 20% of their dry cell weight; and (ii) produce carotenoid(s)at a level at least about 1%, and in some embodiments at least about3-20%, of their dry cell weight, are generated through manipulation ofhost cells (i.e., strains, including, e.g., naturally-occurring strains,strains which have been previously modified, etc.). These manipulatedhost cells are then used to produce carotenoids, so that carotenoidsthat partition into the lipid bodies can readily be isolated.

In general, it will be desirable to balance oleaginy and carotenoidproduction in inventive cells such that, as soon as a minimum desirablelevel of oleaginy is achieved, substantially all further carbon which iscapable of being utilized and diverted into biosynthesis of products isdiverted into a carotenoid production pathway. In some embodiments ofthe invention, this strategy involves engineering cells to beoleaginous; in other embodiments, it involves engineering cells toaccumulate a higher level of lipid, particularly cytoplasmic lipid, thanthey would accumulate in the absence of such engineering even though theengineered cells may not become “oleaginous” as defined herein. In otherembodiments, the extent to which an oleaginous host cell accumulateslipid is actually reduced so that remaining carbon can be utilized incarotenoid production.

Host Cells

Those of ordinary skill in the art will readily appreciate that avariety of yeast and fungal strains exist that are naturally oleaginousor that naturally produce carotenoids. Any of such strains may beutilized as host strains according to the present invention, and may beengineered or otherwise manipulated to generate inventive oleaginous,carotenoid-producing strains. Alternatively, strains that naturally areneither oleaginous nor carotenoid-producing may be employed.Furthermore, even when a particular strain has a natural capacity foroleaginy or for carotenoid production, its natural capabilities may beadjusted as described herein, so as to change the production level oflipid and/or carotenoid. In certain embodiments engineering ormanipulation of a strain results in modification of a type of lipidand/or carotenoid which is produced. For example, a strain may benaturally oleaginous and/or carotenogenic, however engineering ormodification of the strain may be employed so as to change the type oflipid which is accumulated and or to change the type of carotenoid whichis produced.

When selecting a particular yeast or fungal strain for use in accordancewith the present invention, it will generally be desirable to select onewhose cultivation characteristics are amenable to commercial scaleproduction. For example, it will generally (though not necessarilyalways) be desirable to avoid filamentous organisms, or organisms withparticularly unusual or stringent requirements for growth conditions.However, where conditions for commercial scale production can be appliedwhich allow for utilization of filamentous organisms, these may beselected as host cells. In some embodiments of the invention, it will bedesirable to utilize edible organisms as host cells, as they mayoptionally be formulated directly into food or feed additives, or intonutritional supplements, as desired. For ease of production, someembodiments of the invention utilize host cells that are geneticallytractable, amenable to molecular genetics (e.g., can be efficientlytransformed, especially with established or available vectors;optionally can incorporate and/or integrate multiple genes, for examplesequentially; and/or have known genetic sequence; etc), devoid ofcomplex growth requirements (e.g., a necessity for light), mesophilic(e.g., prefer growth temperatures with in the range of about 25-32° C.),able to assimilate a variety of carbon and nitrogen sources and/orcapable of growing to high cell density. Alternatively or additionally,various embodiments of the invention utilize host cells that grow assingle cells rather than multicellular organisms (e.g., as mycelia).

In general, when it is desirable to utilize a naturally oleaginousorganism in accordance with the present invention, any modifiable andcultivatable oleaginous organism may be employed. In certain embodimentsof the invention, yeast or fungi of genera including, but not limitedto, Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces,Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula,Trichosporon, and Yarrowia are employed. In certain particularembodiments, organisms of species that include, but are not limited to,Blakeslea trispora, Candida pulcherrima, C. revkaufi, C. tropicalis,Cryptococcus curvatus, Cunninghamella echinulata, C. elegans, C.japonica, Lipomyces starkeyi, L. lipoferus, Mortierella alpina, M.isabellina, M ramanniana, M vinacea, Mucor circinelloides, Phycomycesblakesleanus, Pythium irregulare, Rhodosporidium torulo ides,Rhodotorula glutinis, R. gracilis, R. graminis, R. mucilaginosa, R.pinicola, Trichosporon pullans, T. cutaneum, and Yarrowia lipolytica areused.

Of these naturally oleaginous strains, some also naturally producecarotenoids and some do not. In most cases, only low levels (less thanabout 0.05% dry cell weight) of carotenoids are produced bynaturally-occurring carotenogenic, oleaginous yeast or fungi. Higherlevels of β-carotene are sometimes produced, but high levels of othercarotenoids are generally not observed.

In general, any organism that is naturally oleaginous andnon-carotenoid-producing (e.g., produce less than about 0.05% dry cellweight, do not produce the carotenoid of interest) may be utilized as ahost cell in accordance with the present invention. In some embodiments,the organism is a yeast or fungus from a genus such as, but not limitedto, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella,Pythium, Trichosporon, and Yarrowia; in some embodiments, the organismis of a species including, but not limited to, Mortierella alpina andYarrowia lipolytica.

Comparably, the present invention may utilize any naturally oleaginous,carotenoid-producing organism as a host cell. In general, the presentinvention may be utilized to increase carbon flow into the isoprenoidpathway in naturally carotenoid-producing organisms (particularly fororganisms other than Blakeslea and Phycomyces), and/or to shiftproduction from one carotenoid (e.g., β-carotene) to another (e.g.,astaxanthin). Introduction of one or more carotenogenic modifications(e.g., increased expression of one or more endogenous or heterologouscarotenogenic polypeptides), in accordance with the present invention,can achieve these goals.

In certain embodiments of the invention, the utilized oleaginous,carotenoid-producing organism is a yeast or fungus, for example of agenus such as, but not limited to, Blakeslea, Mucor, Phycomyces,Rhodosporidium, and Rhodotorula; in some embodiments, the organism is ofa species such as, Mucor circinelloides and Rhodotorula glutinis.

When it is desirable to utilize strains that are naturallynon-oleaginous as host cells in accordance with the present invention,genera of non-oleaginous yeast or fungi include, but are not limited to,Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces,Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces,Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia); in someembodiments, the organism is of a species including, but not limited to,Aspergillus nidulans, A. niger, A. terreus, Botrytis cinerea, Cercosporanicotianae, Fusarium fujikuroi (Gibberella zeae), Kluyveromyces lactis,K. lactis, Neurospora crassa, Pichia pastoris, Puccinia distincta,Saccharomyces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, andXanthophyllomyces dendrorhous (Phaffia rhodozyma).

It will be appreciated that the term “non-oleaginous”, as used herein,encompasses both strains that naturally have some ability to accumulatelipid, especially cytoplasmically, but do not do so to a levelsufficient to qualify as “oleaginous” as defined herein, as well asstrains that do not naturally have any ability to accumulate extralipid, e.g., extra-membranous lipid. It will further be appreciatedthat, in some embodiments of the invention, it will be sufficient toincrease the natural level of oleaginy of a particular host cell, evenif the modified cell does not qualify as oleaginous as defined herein.

As with the naturally oleaginous organisms, some of the naturallynon-oleaginous fungi naturally produce carotenoids, whereas others donot. Genera of naturally non-oleaginous fungi that do not naturallyproduce carotenoids (e.g., produce less than about 0.05% dry cellweight, do not produce carotenoid of interest) may desirably be used ashost cells in accordance with the present invention include, but are notlimited to, Aspergillus, Kluyveromyces, Penicillium, Saccharomyces, andPichia; species include, but are not limited to, Aspergillus niger andSaccharomyces cerevisiae. Genera of naturally non-oleaginous fungi thatdo naturally produce carotenoids and that may desirably be used as hostcells in accordance with the present invention include, but are notlimited to, Botrytis, Cercospora, Fusarium (Gibberella), Neurospora,Puccinia, Sclerotium, Trichoderma, and Xanthophyllomyces (Phaffia);species include, but are not limited to, Xanthophyllomyces dendrorhous(Phaffia rhodozyma).

As discussed above, any of a variety of organisms may be employed ashost cells in accordance with the present invention. In certainembodiments of the invention, host cells will be Yarrowia lipolyticacells. Advantages of Y. lipolytica include, for example, tractablegenetics and molecular biology, availability of genomic sequence (see,for example. Sherman et al. Nucleic Acids Res. 32 (Databaseissue):D315-8, 2004), suitability to various cost-effective growthconditions, and ability to grow to high cell density. In addition, Y.lipolytica is naturally oleaginous, such that fewer manipulations may berequired to generate an oleaginous, carotenoid-producing Y. lipolyticastrain than might be required for other organisms. Furthermore, there isalready extensive commercial experience with Y. lipolytica.

Saccharomyces cerevisiae is also a useful host cell in accordance withthe present invention, particularly due to its experimental tractabilityand the extensive experience that researchers have accumulated with theorganism. Although cultivation of Saccharomyces under high carbonconditions may result in increased ethanol production, this cangenerally be managed by process and/or genetic alterations.

Additional useful hosts include Xanthophyllomyces dendrorhous (Phaffiarhodozyma), which is experimentally tractable and naturallycarotenogenic. Xanthophyllomyces dendrorhous (Phaffia rhodozyma) strainscan produce several carotenoids, including astaxanthin.

Aspergillus niger and Mortierella alpina accumulate large amounts ofcitric acid and fatty acid, respectively; Mortierella alpina is alsooleaginous.

Neurospora or Gibberella are also useful. They are not naturallyoleaginous and tend to produce very low levels of carotenoids, thusextensive modification may be required in accordance with the presentinvention. Neurospora and Gibberella are considered relatively tractablefrom an experimental standpoint. Both are filamentous fungi, such thatproduction at commercial scales can be a challenge necessary to overcomein utilization of such strains.

Mucor circinelloides is another available useful species. While itsmolecular genetics are generally less accessible than are those of someother organisms, it naturally produces β-carotene, thus may require lessmodification than other species available.

Molecular genetics can be performed in Blakeslea, though significanteffort may be required. Furthermore, cost-effective fermentationconditions can be challenging, as, for example, it may be required thatthe two mating types are mixed. Fungi of the genus Phycomyces are alsopossible sources which have the potential to pose fermentation processchallenges, and these fungi are also may be less amenable to manipulatethan several other potential host organisms.

Those of ordinary skill in the art will appreciate that the selection ofa particular host cell for use in accordance with the present inventionwill also affect, for example, the selection of expression sequencesutilized with any heterologous polypeptide to be introduced into thecell, and will also influence various aspects of culture conditions,etc. Much is known about the different gene regulatory requirements,protein targeting sequence requirements, and cultivation requirements,of different host cells to be utilized in accordance with the presentinvention (see, for example, with respect to Yarrowia, Barth et al. FEMSMicrobiol Rev. 19:219, 1997; Madzak et al. J. Biotechnol. 109:63, 2004;see, for example, with respect to Xanthophyllomyces, Verdoes et al. ApplEnviron Microbiol 69: 3728-38, 2003; Visser et al. FEMS Yeast Res 4:221-31, 2003; Martinez et al. Antonie Van Leeuwenhoek. 73(2):147-53,1998; Kim et al. Appl Environ Microbiol. 64(5):1947-9, 1998; Wery et al.Gene. 184(1):89-97, 1997; see, for example, with respect toSaccharomyces, Guthrie and Fink Methods in Enzymology 194:1-933, 1991).In certain aspects, for example, targeting sequences of the host cell(or closely related analogs) may be useful to include for directingheterologous proteins to subcellular localization. Thus, such usefultargeting sequences can be added to heterologous sequence for properintracellular localization of activity. In other aspects (e.g., additionof mitochondrial targeting sequences), heterologous targeting sequencesmay be eliminated or altered in the selected heterologous sequence(e.g., alteration or removal of source organism plant chloroplasttargeting sequences).

Engineering Oleaginy

All living organisms synthesize lipids for use in their membranes andvarious other structures. However, most organisms do not accumulate inexcess of about 10% of their dry cell weight as total lipid, and most ofthis lipid generally resides within cellular membranes.

Significant biochemical work has been done to define the metabolicenzymes necessary to confer oleaginy on microorganisms (primarily forthe purpose of engineering single cell oils as commercial sources ofarachidonic acid and docosahexaenoic acid; see for example RatledgeBiochimie 86:807, 2004, the entire contents of which are incorporatedherein by reference). Although this biochemical work is compelling,prior to the present invention, there have been no reports of de novooleaginy being established through genetic engineering with the genesencoding the key metabolic enzymes.

It should be noted that oleaginous organisms typically only accumulatelipid when grown under conditions of carbon excess and nitrogen or othernutrient limitation. Under these conditions, the organism readilydepletes the limiting nutrient but continues to assimilate the carbonsource. The “excess” carbon is channeled into lipid biosynthesis so thatlipids (usually triacylglycerols) accumulate in the cytosol, typicallyin the form of bodies.

In general, it is thought that, in order to be oleaginous, an organismmust produce both acetyl-CoA and NADPH in the cytosol, which can then beutilized by the fatty acid synthase machinery to generate lipids. In atleast some oleaginous organisms, acetyl-CoA is generated in the cytosolthrough the action of ATP-citrate lyase, which catalyzes the reaction:

citrate+CoA+ATP→acetyl-CoA+oxaloacetate+ADP+P_(i).  (1)

Of course, in order for ATP-citrate lyase to generate appropriate levelsof acetyl-CoA in the cytosol, it must first have an available pool ofits substrate citric acid. Citric acid is generated in the mitochondriaof all eukaryotic cells through the tricarboxylic acid (TCA) cycle, andcan be moved into the cytosol (in exchange for malate) by citrate/malatetranslocase.

In most oleaginous organisms, and in some non-oleaginous organisms, theenzyme isocitrate dehydrogenase, which operates as part of the TCA cyclein the mitochondria, is strongly AMP-dependent. Thus, when AMP isdepleted from the mitochondria, this enzyme is inactivated. Whenisocitrate dehydrogenase is inactive, isocitrate accumulates in themitochondria. This accumulated isocitrate is then equilibrated withcitric acid, presumably through the action of aconitase. Therefore,under conditions of low AMP, citrate accumulates in the mitochondria. Asnoted above, mitochondrial citrate is readily transported into thecytosol.

AMP depletion, which in oleaginous organisms is believed to initiate thecascade leading to accumulation of citrate (and therefore acetyl-CoA) inthe cytoplasm, occurs as a result of the nutrient depletion mentionedabove. When oleaginous cells are grown in the presence of excess carbonsource but under conditions limiting for nitrogen or some othernutrient(s), the activity of AMP deaminase, which catalyzes thereaction:

AMP→inosine 5′-monophosphate+NH₃  (2)

is strongly induced. The increased activity of this enzyme depletescellular AMP in both the cytosol and the mitochondria. Depletion of AMPfrom the mitochondria is thought to inactivate the AMP-dependentisocitrate dehydrogenase, resulting in accumulation of citrate in themitochondria and, therefore, the cytosol. This series of events isdepicted diagrammatically in FIG. 2.

As noted above, oleaginy requires both cytosolic acetyl-CoA andcytosolic NADPH. It is believed that, in many oleaginous organisms,appropriate levels of cytosolic NADPH are provided through the action ofmalic enzyme (Enzyme 3 in FIG. 2). Some oleaginous organisms (e.g.,Lipomyces and some Candida) do not appear to have malic enzymes,however, so apparently other enzymes can provide comparable activity,although it is expected that a dedicated source of NADPH is probablyrequired for fatty acid synthesis (see, for example, Wynn et al.,Microbiol 145:1911, 1999; Ratledge Adv. Appl. Microbiol. 51:1, 2002,each of which is incorporated herein by reference in its entirety).

Thus, according to the present invention, the oleaginy of a hostorganism may be enhanced by modifying the expression or activity of oneor more polypeptides involved in generating cytosolic acetyl-CoA and/orNADPH. For example, modification of the expression or activity of one ormore of acetyl-CoA carboxylase, pyruvate decarboxylase, isocitratedehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminase canenhance oleaginy in accordance with the present invention. Exemplarypolypeptides which can be utilized or derived so as to enhance oleaginyin accordance with the present invention include, but are not limited tothose acetyl-CoA carboxylase, pyruvate decarboxylase, isocitratedehydrogenase, ATP-citrate lyase, malic enzyme, and AMP-deaminasepolypeptides provided in Table 1, Table 2, Table 3, Table 4, Table 5,and Table 6, respectively.

In some embodiments of the invention, where an oleaginous host cell isemployed, enzymes and regulatory components relevant to oleaginy arealready in place but could be modified, if desired, by for examplealtering expression or activity of one or more oleaginic polypeptidesand/or by introducing one or more heterologous oleaginic polypeptides.In those embodiments of the invention where a non-oleaginous host cellis employed, it is generally expected that at least one or moreheterologous oleaginic polypeptides will be introduced.

The present invention contemplates not only introduction of heterologousoleaginous polypeptides, but also adjustment of expression or activitylevels of heterologous or endogenous oleaginic polypeptides, including,for example, alteration of constitutive or inducible expressionpatterns. In some embodiments of the invention, expression patterns areadjusted such that growth in nutrient-limiting conditions is notrequired to induce oleaginy. For example, genetic modificationscomprising alteration and/or addition of regulatory sequences (e.g.,promoter elements, terminator elements) may be utilized to conferparticular regulation of expression patterns. Such genetic modificationsmay be utilized in conjunction with endogenous genes (e.g., forregulation of endogenous oleagenic polypeptide(s)); alternatively, suchgenetic modifications may be included so as to confer regulation ofexpression of at least one heterologous polypeptide (e.g., oleagenicpolypeptide(s)). For example, promoters including, but not limited toTef1, Gpd1 promoters can be used in conjunction with endogenous genesand/or heterolous genes for modification of expression patterns ofendogenous oleaginic polypeptides and/or heterolous oleagenicpolypeptides. Similarly, exemplary terminator sequences include, but arenot limited to, use of Y. lipolytica XPR2 terminator sequences.

In some embodiments, at least one oleaginic polypeptide is introducedinto a host cell. In some embodiments of the invention, a plurality(e.g., two or more) of different oleaginic polypeptides is introducedinto the same host cell. In some embodiments, the plurality of oleaginicpolypeptides contains polypeptides from the same source organism; inother embodiments, the plurality includes polypeptides independentlyselected from different source organisms.

Representative examples of a variety of oleaginic polypeptides that maybe introduced into or modified within host cells according to thepresent invention, include, but are not limited to, those provided inTables 1-6. As noted above, it is expected that at least some of thesepolypeptides (e.g., malic enzyme and ATP-citrate lyase) should desirablyact in concert, and possibly together with one or more components offatty acid synthase, such that, in some embodiments of the invention, itwill be desirable to utilize two or more oleaginic polypeptides from thesame source organism.

In general, source organisms for oleaginic polypeptides to be used inaccordance with the present invention include, but are not limited to,Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces,Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidium, Rhodotorula,Trichosporon, Yarrowia, Aspergillus, Botrytis, Cercospora, Fusarium(Gibberella), Kluyveromyces, Neurospora, Penicillium, Pichia(Hansenula), Puccinia, Saccharomyces, Sclerotium, Trichoderma, andXanthophyllomyces (Phaffia). In some embodiments, the source species foracetyl CoA carboxylase, ATP-citrate lyase, malice enzyme and/or AMPdeaminase polypeptides include, but are not limited to, Aspergillusnidulans, Cryptococcus neoformans, Fusarium fujikuroi, Kluyveromyceslactis, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomycespombe, Ustilago maydis, and Yarrowia lipolytica; in some embodiments,source species for pyruvate decarboxylase or isocitrate dehydrogenasepolypeptides include, but are not limited to Neurospora crassa,Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Aspergillus niger,Saccharomyces cerevisiae, Mucor circinelloides, Rhodotorula glutinis,Candida utilis, Mortierella alpina and Yarrowia

Engineering Carotenoid Production

Carotenoids are synthesized from isoprenoid precursors, some of whichare also involved in the production of steroids and sterols. The mostcommon isoprenoid biosynthesis pathway, sometimes referred to as the“mevalonate pathway”, is generally depicted in FIG. 3. As shown,acetyl-CoA is converted, via hydroxymethylglutaryl-CoA (HMG-CoA), intomevalonate. Mevalonate is then phosphorylated and converted into thefive-carbon compound isopentenyl pyrophosphate (IPP). Followingisomerization of IPP into dimethylallyl pyrophosphate (DMAPP), threesequential condensation reactions with additional molecules of IPPgenerate the ten-carbon molecule geranyl pyrophosphate (GPP), followedby the fifteen-carbon molecule farnesyl pyrophosphate (FPP), and finallythe twenty-carbon compound geranylgeranyl pyrophosphate (GGPP).

An alternative isoprenoid biosynthesis pathway, that is utilized by someorganisms (particularly bacteria) and is sometimes called the“mevalonate-independent pathway”, is depicted in FIG. 4. This pathway isinitiated by the synthesis of 1-deoxy-D-xyloglucose-5-phosphate (DOXP)from pyruvate and glyceraldehyde-3-phosphate. DOXP is then converted,via a series of reactions shown in FIG. 4, into IPP, which isomerizesinto DMAPP and is then converted, via GPP and FPP, into GGPP as shown inFIG. 3 and discussed above.

Various proteins involved in isoprenoid biosynthesis have beenidentified and characterized in a number of organisms. Moreover, variousaspects of the isoprenoid biosynthesis pathway are conserved throughoutthe fungal, bacterial, plant and animal kingdoms. For example,polypeptides corresponding to the acetoacetyl-CoA thiolase, HMG-CoAsynthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonatekinase, mevalonate pyrophosphate decarboxylase, IPP isomerase, FPPsynthase, and GGPP synthase shown in FIG. 3 have been identified in andisolated from a wide variety of organisms and cells. Representativeexamples of a wide variety of such polypeptides are provided in Tables7-15. One or more of the polypeptides selected from those provided inany one of Tables 7-15 may be utilized or derived for use in the methodsand compositions in accordance with the present invention.

According to the present invention, carotenoid production in a hostorganism may be adjusted by modifying the expression or activity of oneor more proteins involved in isoprenoid biosynthesis. In someembodiments, such modification involves introduction of one or moreheterologous isoprenoid biosynthesis polypeptides into the host cell;alternatively or additionally, modifications may be made to theexpression or activity of one or more endogenous or heterologousisoprenoid biosynthesis polypeptides. Given the considerableconservation of components of the isoprenoid biosynthesis polypeptides,it is expected that heterologous isoprenoid biosynthesis polypeptideswill often function even in significantly divergent organisms.Furthermore, should it be desirable to introduce more than oneheterologous isoprenoid biosynthesis polypeptide, in many casespolypeptides from different source organisms will function together. Insome embodiments of the invention, a plurality of different heterologousisoprenoid biosynthesis polypeptides is introduced into the same hostcell. In some embodiments, this plurality contains only polypeptidesfrom the same source organism (e.g., two or more sequences of, orsequences derived from, the same source organism); in other embodimentsthe plurality includes polypeptides independently selected from fromdifferent source organisms (e.g., two or more sequences of, or sequencesderived from, at least two independent source organisms).

In some embodiments of the present invention that utilize heterologousisoprenoid biosynthesis polypeptides, the source organisms include, butare not limited to, fungi of the genera Blakeslea, Candida,Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces,Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yarrowia,Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), Kluyveromyces,Neurospora, Penicillium, Pichia (Hansenula), Puccinia, Saccharomyces,Schizosaccharomyces, Sclerotium, Trichoderms, Ustilago, andXanthophyllomyces (Phaffia). In certain embodiments, the sourceorganisms are of a species including, but not limited to, Cryptococcusneoformans, Fusarium fujikuroi, Kluyverimyces lactis, Neurospora crassa,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Ustilago maydis,and Yarrowia lipolytica.

As noted above, the isoprenoid biosynthesis pathway is also involved inthe production of non-carotenoid compounds, such as sterols, steroids,and vitamins, such as vitamin E or vitamin K. Proteins that act onisoprenoid biosynthesis pathway intermediates, and divert them intobiosynthesis of non-carotenoid compounds are therefore indirectinhibitors of carotenoid biosynthesis (see, for example, FIG. 5, whichillustrates points at which isoprenoid intermediates are channeled intoother biosynthesis pathways). Such proteins are therefore consideredisoprenoid biosynthesis competitor polypeptides. Reductions of the levelor activity of such isoprenoid biosynthesis competitor polypeptides areexpected to increase carotenoid production in host cells according tothe present invention.

In some embodiments of the present invention, production or activity ofendogenous isoprenoid biosynthesis competitor polypeptides may bereduced or eliminated in host cells. In some embodiments, this reductionor elimination of the activity of an isoprenoid biosynthesis competitorpolypeptide can be achieved by treatment of the host organism with smallmolecule inhibitors of enzymes of the ergosterol biosynthetic pathway.Enzymes of the ergosterol biosynthetic pathway include, for example,squalene synthase, squalene epoxidase, 2,3-oxidosqualene-lanosterolcyclase, cytochrome P450 lanosterol 14α-demethylase, C-14 sterolreductase, C-4 sterol methyl oxidase, SAM:C-24 sterol methyltransferase,C-8 sterol isomerase, C-5 sterol desaturase, C-22 sterol desaturase, andC-24 sterol reductase. Each of these enzymes is considered an isoprenoidbiosynthesis competitor polypeptide. Regulators of these enzymes mayalso be considered isoprenoid biosynthesis competitor polypeptides(e.g., the yeast proteins Sut1 (Genbank Accession JC4374 GI:2133159) andMot3 (Genbank Accession NP_(—)013786 GI:6323715), which may or may nothave homologs in other organisms.

In other embodiments, reduction or elimination of the activity of anisoprenoid biosynthesis competitor polypeptide can be achieved bydecreasing activity of the ubiquinone biosynthetic pathway. Thecommitment step in ubiquinone biosynthesis is the formation ofpara-hydroxybenzoate (PHB) from tyrosine or phenylalanine in mammals orchorismate in bacteria, followed by condensation of PHB and isopreneprecursor, resulting in addition of the prenyl group. This reaction iscatalyzed by PHB-polyprenyltransferase. The isoprenoid side chain ofubiquinone is determined by the prenyldiphosphate synthase enzyme. The3-decaprenyl-4-hydroxybenzoic acid resulting from the condensation ofPHB and decaprenyldiphosphate reaction undergoes further modifications,which include hydroxylation, methylation and decarboxylation, in orderto form ubiquinone (CoQ10). Thus, inhibition of prenyldiphosphatesynthase leading from farnesyldiphosphate to extended isoprenoids, orinhibition of PHB polyprenyltransferase may be useful in increasing theamount of isoprenoid available for carotenoid biosynthesis. (Examples ofprenyldiphosphate synthase and PHB-polyprenyltransferase enzymes aredepicted in Tables 29 and 30, respectively).

Known small molecule inhibitors of isoprenoid biosynthesis competitorenzymes include, but are not limited to, zaragosic acid (includinganalogs thereof such as TAN1607A (Biochem Biophys Res Commun 1996 Feb.15; 219(2):515-520)), RPR 107393(3-hydroxy-3-[4-(quinolin-6-yl)phenyl]-1-azabicyclo[2-2-2]octanedihydrochloride; J Pharmacol Exp Ther. 1997 May; 281(2):746-52),ER-28448(5-{N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino}-1,1-penthylidenebis(phosphonicacid) trisodium salt; Journal of Lipid Research, Vol. 41, 1136-1144,July 2000), BMS-188494 (The Journal of Clinical Pharmacology, 1998;38:1116-1121), TAK-475 (1-[2-[(3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl]acetyl]piperidin-4-aceticacid; Eur J. Pharmacol. 2003 Apr. 11; 466(1-2):155-61), YM-53601((E)-2-[2-fluoro-2-(quinuclidin-3-ylidene) ethoxy]-9H-carbazolemonohydrochloride; Br J. Pharmacol. 2000 September; 131(1):63-70), orsqualestatin I that inhibit squalene synthase; terbinafine that inhibitssqualene epoxidase; various azoles that inhibit cytochrome P450lanosterol 14α-demethylase; and fenpropimorph that inhibits the C-14sterol reductase and the C-8 sterol isomerase. In other embodiments,heterologous isoprenoid biosynthesis competitor polypeptides may beutilized (whether functional or non-functional; in some embodiments,dominant negative mutants are employed).

One particular isoprenoid biosynthesis competitor polypeptide usefulaccording to the present invention is squalene synthase which has beenidentified and characterized from a variety of organisms; representativeexamples of squalene synthase polypeptide sequences are included inTable 16. In some embodiments of the invention that utilize squalenesynthase (or modifications of squalene synthase) source organismsinclude, but are not limited to, Neurospora crassa, Xanthophyllomycesdendrorhous (Phaffia rhodozyma), Aspergillus niger, Saccharomycescerevisiae, Mucor circinelloides, Rhotorula glutinis, Candida utilis,Mortierella alpina, and Yarrowia lipolytica.

The carotenoid biosynthesis pathway branches off from the isoprenoidbiosynthesis pathway at the point where GGPP is formed. The commitmentstep in carotenoid biosynthesis is the formation of phytoene by thehead-to-head condensation of two molecules of GGPP, catalyzed byphytoene synthase (often called crtB; see FIG. 6). A series ofdehydrogenation reactions, each of which increases the number ofconjugated double bonds by two, converts phytoene into lycopene vianeurosporene. The pathway branches at various points, both before andafter lycopene production, so that a wide range of carotenoids can begenerated. For example, action of a cyclase enzyme on lycopene generatesγ-carotene; action of a desaturase instead produces3,4-didehydrolycopene. γ-carotene is converted to β-carotene through theaction of a cyclase. β-carotene can be processed into any of a number ofproducts (see, for example, FIG. 6C), including astaxanthin (viaechinone, hydroxyechinone, and phoenicoxanthin).

According to the present invention, carotenoid production in a hostorganism may be adjusted by modifying the expression or activity of oneor more proteins involved in carotenoid biosynthesis. As indicated, insome embodiments, it will be desirable to utilize as host cellsorganisms that naturally produce one or more carotenoids. In some suchcases, the focus will be on increasing production of anaturally-produced carotenoid, for example by increasing the leveland/or activity of one or more proteins involved in the synthesis ofthat carotenoid and/or by decreasing the level or activity of one ormore proteins involved in a competing biosynthetic pathway.Alternatively or additionally, in some embodiments it will be desirableto generate production of one or more carotenoids not naturally producedby the host cell.

According to some embodiments of the invention, it will be desirable tointroduce one or more heterologous carotenogenic polypeptides into ahost cell. As will be apparent to those of ordinary skill in the art,any of a variety of heterologous polypeptides may be employed; selectionwill consider, for instance, the particular carotenoid whose productionis to be enhanced. The present invention contemplates not onlyintroduction of heterologous carotenogenic polypeptides, but alsoadjustment of expression or activity levels of heterologous orendogenous carotenogenic polypeptides, including, for example,alteration of constitutive or inducible expression patterns. In someembodiments of the invention, expression patterns are adjusted such thatgrowth in nutrient-limiting conditions is not required to induceoleaginy. For example, genetic modifications comprising alterationand/or addition of regulatory sequences (e.g., promoter elements,terminator elements) may be utilized to confer particular regulation ofexpression patterns. Such genetic modifications may be utilized inconjunction with endogenous genes (e.g., for regulation of endogenouscarotenogenic); alternatively, such genetic modifications may beincluded so as to confer regulation of expression of at least oneheterologous polypeptide (e.g., carotenogenic polypeptide(s)). Forexample, promoters including, but not limited to Tef1, Gpd1 promoterscan be used in conjunction with endogenous genes and/or heterolous genesfor modification of expression patterns of endogenous carotenogenicpolypeptide(s) and/or heterolous carotenogenic polypeptide(s).Similarly, exemplary terminator sequences include, but are not limitedto, use of Y. lipolytica XPR2 terminator sequences.

As indicated in FIG. 6 and in the literature, proteins involved incarotenoid biosynthesis include, but are not limited to, phytoenesynthase, phytoene dehydrogenase, lycopene cyclase, carotenoid ketolase,carotenoid hydroxylase, astaxanthin synthase (a single multifunctionalenzyme found in some source organisms that typically has both ketolaseand hydroxylase activities), carotenoid epsilon hydroxylase, lycopenecyclase (beta and epsilon subunits), carotenoid glucosyltransferase, andacyl CoA:diacyglycerol acyltransferase. Representative example sequencesfor these carotenoid biosynthesis polypeptides are provided in Tables17-25.

Xanthophylls can be distinguished from other carotenoids by the presenceof oxygen containing functional groups on their cyclic end groups. Forinstance, lutein and zeaxanthin contain a single hydroxyl group on eachof their terminal ring structures, while astaxanthin contains both aketo group and a hydroxyl on each terminal ring. This property makesxanthophylls more polar than carotenes such as beta-carotene andlycopene, and thus dramatically reduces their solubility in fats andlipids. Naturally occurring xanthophylls are often found as esters ofthe terminal hydroxyl groups, both mono- and diesters of fatty acids.They also occur as glucosides in certain species of bacteria. Thesolubility and dispersibility of xanthophylls can be greatly modified bythe addition of ester moieties, and it is known that esterification canalso affect the absorbability and/or bioavailability of a givencarotenoid. It is an objective of this invention to maximize the amountof a particular xanthophyll accumulating within the intracellulartriacylglyceride fraction of oleaginous yeasts, and one mechanism forachieving this goal is to increase the hydrophobic nature of thexanthophyll product that accumulates. One way of achieving this is toengineer the production of fatty-acyl mono- and/or diesters of thetarget xanthophyll compound.

A variety of enzymes can function to esterify carotenoids. For example,carotenoid glucosyltransferases have been identified in severalbacterial species (see, e.g., Table 24). In addition, acylCoA:diacyglycerol acyltransferase (DGAT) and acyl CoA:monoacylglycerolacyltransferases (MGAT), which function in the final steps oftriacylglycerol biosynthesis, are likely to serve an additional role inthe esterification of xanthophylls. Representative DGAT polypeptides areshown in Table 25. Furthermore, other enzymes may specifically modifycarotenoids and molecules of similar structure (e.g. sterols) and beavailable for modification and ester production.

In some embodiments of the invention, potential source organisms forcarotenoid biosynthesis polypeptides include, but are not limited to,genera of naturally oleaginous or non-oleaginous fungi that naturallyproduce carotenoids. These include, but are not limited to, Botrytis,Cercospora, Fusarium (Gibberella), Mucor, Neurospora, Phycomyces,Puccina, Rhodotorula, Sclerotium, Trichoderma, and Xanthophyllomyces.Exemplary species include, but are not limited to, Neurospora crassa,Xanthophyllomyces dendrorhous (Phaffia rhodozyma), Mucor circinelloides,and Rhodotorula glutinis. Of course, carotenoids are produced by a widerange of diverse organisms such as plants, algae, yeast, fungi,bacteria, cyanobacteria, etc. Any such organisms may be source organismsfor carotenoid biosynthesis polypeptides according to the presentinvention.

It will be appreciated that the particular carotenogenic modification tobe applied to a host cell in accordance with the present invention willbe influenced by which carotenoid(s) is desired to be produced. Forexample, isoprenoid biosynthesis polypeptides are relevant to theproduction of most carotenoids. Carotenoid biosynthesis polypeptides arealso broadly relevant. Ketolase is particularly relevant for productionof canthaxanthin, as hydroxylase is for production of lutein andzeaxanthin, among others. Both hydroxylase and ketolase (or astaxanthinsynthase) are particularly useful for production of astaxanthin.

Production and Isolation of Carotenoids

As discussed above, accumulation of lipid bodies in oleaginous organismsis generally induced by growing the relevant organism in the presence ofexcess carbon source and limiting nitrogen. Specific conditions forinducing such accumulation have previously been established for a numberof different oleaginous organisms (see, for example, Wolf (ed.)Nonconventional yeasts in biotechnology Vol. 1, Springer-Verlag, Berlin,Germany, pp. 313-338; Lipids 18(9):623, 1983; Indian J. Exp. Biol.35(3):313, 1997; J. Ind. Microbial. Biotechnol. 30(1):75, 2003;Bioresour Technol. 95(3):287, 2004, each of which is incorporated hereinby reference in its entirety).

In general, it will be desirable to cultivate inventive modified hostcells under conditions that allow accumulation of at least about 20% oftheir dry cell weight as lipid. In other embodiments, the inventivemodified host cells are grown under conditions that permit accumulationof at least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or even 80% or more oftheir dry cell weight as lipid. In certain embodiments, the host cellsutilized are cells which are naturally oleaginous, and induced toproduce lipid to the desired levels. In other embodiments, the hostcells are cells which naturally produce lipid, but have been engineeredto increase production of lipid such that desired levels of lipidproduction and accumulation are achieved.

In certain embodiments, the host cells of the invention are notnaturally oleaginous, but have been engineered to produce lipid suchthat desired levels of lipid production are obtained. Those of ordinaryskill in the art will appreciate that, in general, growth conditionsthat are effective for inducing lipid accumulation in a source organism,may well also be useful for inducing lipid accumulation in a host cellinto which the source organism's oleaginic polypeptides have beenintroduced. Of course, modifications may be required in light ofcharacteristics of the host cell, which modifications are within theskill of those of ordinary skill in the art.

It will also be appreciated by those of ordinary skill in the art thatit will generally be desirable to ensure that production of the desiredcarotenoid by the inventive modified host cell occurs at an appropriatetime in relation to the induction of oleaginy such that thecarotenoid(s) accumulate(s) in the lipid bodies. In some embodiments, itwill be desirable to induce production of the carotenoid(s) in a hostcell which does not naturally produce the carotenoid(s), such thatdetectable levels of the carotenoid(s) is/are produced. In certainaspects the host cells which do not naturally produce a certaincarotenoid(s) are capable of production of other carotenoid(s) (e.g.certain host cells may, for example, naturally produce β-carotene butmay not naturally produce astaxanthin); in other aspects the host cellsdo not naturally produce any carotenoid(s). In other embodiments, itwill be desirable to increase production levels of carotenoid(s) in ahost cell which does naturally produce low levels of the carotenoid(s),such that increased detectable levels of the carotenoid(s) are produced.In certain aspects, the host cells which do naturally produce thecarotenoid(s) (e.g., β-carotene) also produce additional carotenoid(s)(e.g., astaxanthin, etc.); in still other aspects, the cells whichnaturally produce the carotenoid(s) (e.g., β-carotene) do not produceadditional carotenoid(s).

In certain embodiments of the invention, it will be desirable toaccumulate carotenoids to levels (i.e., considering the total amount ofall produced carotenoids together) that are greater than at least about1% of the dry weight of the cells. In some embodiments, the totalcarotenoid accumulation in the lipid bodies will be to a level at leastabout 2%, at least about 3%, at least about 4%, at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%,at least about 10%, at least about 11%, at least about 12%, at leastabout 13%, at least about 14%, at least about 15%, at least about 16%,at least about 17%, at least about 18%, at least about 19%, at leastabout 20% or more of the total dry weight of the cells. In certainembodiments of the invention, it will be desirable to achieve totallevels of carotenoid accumulation in the lipid bodies (i.e., consideringthe total amount of all produced carotenoids together) that are greaterthan at least about 1% of the dry weight of the cells. In someembodiments, the total carotenoid accumulation in the lipid bodies willbe to a level at least about 2%, at least about 3%, at least about 4%,at least about 5%, at least about 6%, at least about 7%, at least about8%, at least about 9%, at least about 10%, at least about 11%, at leastabout 12%, at least about 13%, at least about 14%, at least about 15%,at least about 16%, at least about 17%, at least about 18%, at leastabout 19%, at least about 20% or more of the total dry weight of thecells.

Bacterial carotenogenic genes have already been demonstrated to betransferrable to other organisms, and are therefore particularly usefulin accordance with the present invention (see, for example, Miura etal., Appl. Environ. Microbiol. 64:1226, 1998). In other embodiments, itmay be desirable to utilize genes from other source organisms such asplant, alga, or microalgae; these organisms provide a variety ofpotential sources for ketolase and hydroxylase polypeptides. Stilladditional useful source organisms include fungal, yeast, insect,protozoal, and mammalian sources of polypeptides.

In certain embodiments, the Mucor circinelloides multi-functionalphytoene synthase/lycopene cyclase and the Neurospora crassa phytoenedehydrogenase genes can be expressed in Yarrowia lipolytica. Subsequentoverexpression of the catalytic domain from N. crassahydroxymethylglutaryl-CoA reductase and/or treatment of the modified Y.lipolytica strains with the squalene synthase inhibitor zaragozic acidfurther increases carotenoid production. Finally, Paracoccus marcusiigenes encoding carotenoid hydroxylase and carotenoid ketolase enzymesare expressed in Y. lipolytica β-carotene-producing strains, and thismodification results in the accumulation of astaxanthin. Similarapproaches to enhance carotenoid production could be employed in otheroleaginous or non-oleaginous host organisms can be undertaken, using thesame, homologous, or functionally similar carotogenic polypeptides.

It should be noted that, for inventive organisms that produce more thanone carotenoid, it will sometimes be possible to adjust the relativeamounts of individual carotenoids produced by adjusting growthconditions. For example, it has been reported that controlling theconcentration of dissolved oxygen in a culture during cultivation canregulate relative production levels of certain carotenoids such asβ-carotene, echinenone, β-cryptoxanthin, 3-hydroxyechinenone,asteroidenone, canthaxanthin, zeaxanthin, adonirubin, adonixanthin andastaxanthin (see, for example, U.S. Pat. No. 6,825,002 to Tsubokura etal., the entire contents of which are incorporated herein by reference).

Particularly for embodiments of the present invention directed towardproduction of astaxanthin, it will often be desirable to utilize one ormore genes from a natural astaxanthin-producing organism. Where multipleheterologous polypeptides are to be expressed, it may be desirable toutilize the same source organism for all, or to utilize closely relatedsource organisms.

One advantage provided by the present invention is that, in addition toallowing the production of high levels of carotenoids, the presentinvention allows those produced compounds to be readily isolated becausethey accumulate in the lipid bodies within oleaginous organisms. Methodsand systems for isolating lipid bodies have been established for a widevariety of oleaginous organisms (see, for example, U.S. Pat. Nos.5,164,308; 5,374,657; 5,422,247; 5,550,156; 5,583,019; 6,166,231;6,541,049; 6,727,373; 6,750,048; and 6,812,001, each of which isincorporated herein by reference in its entirety). In brief, cells aretypically recovered from culture, often by spray drying, filtering orcentrifugation. In some instances, cells are homogenized and thensubjected to supercritical liquid extraction or solvent extraction(e.g., with solvents such as chloroform, hexane, methylene chloride,methanol, isopropanol, ethyl acetate, etc.), yielding a crude oilsuspension. This oil suspension may optionally be refined as known inthe art. Refined oils may be used directly as feed or food additives.Alternatively or additionally, carotenoids can be isolated from the oilusing conventional techniques.

Given the sensitivity of carotenoids generally to oxidation, manyembodiments of the invention employ oxidative stabilizers (e.g.,tocopherols, vitamin C; ethoxyquin; vitamin E, BHT, BHA, TBHQ, etc, orcombinations thereof) during and/or after carotenoid isolation.Alternatively or additionally, microencapsulation, for example withproteins, may be employed to add a physical barrier to oxidation and/orto improve handling (see, for example, U.S. Patent Application2004/0191365).

Uses

Carotenoids produced according to the present invention can be utilizedin any of a variety of applications, for example exploiting theirbiological or nutritional properties (e.g., anti-oxidant,anti-proliferative, etc.) and/or their pigment properties. For example,according to the present invention, carotenoids may be used inpharmaceuticals (see, for example, Bertram, Nutr. Rev. 57:182, 1999;Singh et al., Oncology 12:1643, 1998; Rock, Pharmacol. Ther. 75:185,1997; Edge et al, J. Photochem Photobiol 41:189, 1997; U.S. PatentApplication 2004/0116514; U.S. Patent Application 2004/0259959), foodsupplements (see, for example, Koyama et al, J. Photochem Photobiol9:265, 1991; Bauernfeind, Carotenoids as colorants and vitamin Aprecursors, Academic Press, NY, 1981; U.S. Patent Application2004/0115309; U.S. Patent Application 2004/0234579), electro-opticapplications, animal feed additives (see, for example, Krinski, PureAppl. Chem. 66:1003, 1994; Polazza et al., Meth. Enzymol. 213:403,1992), cosmetics (as anti-oxidants and/or as cosmetics, includingfragrances; see for example U.S. Patent Application 2004/0127554), etc.Carotenoids produced in accordance with the present invention may alsobe used as intermediates in the production of other compounds (e.g.,steroids, etc.).

For example, astaxanthin and/or esters thereof may be useful in avariety of pharmaceutical applications and health foods includingtreatment of inflammatory diseases, asthma, atopic dermatitis,allergies, multiple myeloma, arteriosclerosis, cardiovascular disease,liver disease, cerebrovascular disease, thrombosis,neoangiogenesis-related diseases, including cancer, rheumatism, diabeticretinopathy; macular degeneration and brain disorder, hyperlipidemia,kidney ischemia, diabetes, hypertension, tumor proliferation andmetastasis; and metabolic disorders. Additionally, carotenoids andastaxanthin may be useful in the prevention and treatment of fatigue,for improving kidney function in nephropathy from inflammatory diseases,as well as prevention and treatment of other life habit-relateddiseases. Still further, astaxanthin has been found to play a role asinhibitors of various biological processes, including interleukininhibitors, phosphodiesterase inhibitors inhibitors, phospholipase A2inhibitors, cyclooxygenase-2 inhibitors, matrix metalloproteinaseinhibitors, capillary endothelium cell proliferation inhibitors,lipoxygenase inhibitors. See, e.g., Japanese Publication No. 2006022121,published 20060126 (JP Appl No. 2005-301156 filed 20051017); JapanesePublication No. 2006016408, published 20060119 (JP Appl No. 2005-301155filed 20051017); Japanese Publication No. 2006016409, published 20060119(JP Appl No. 2005-301157 filed 20051017); Japanese Publication No.2006016407, published 20060119 (JP Appl No. 2005-301153 filed 20051017);Japanese Publication No. 2006008717, published 20060112 (JP Appl No.2005-301151 filed 20051017); Japanese Publication No. 2006008716,published 20060112 (JP Appl No. 2005-301150 filed 20051017); JapanesePublication No. 2006008720, published 20060112 (JP Appl No. 2005-301158filed 20051017); Japanese Publication No. 2006008719, published 20060112(JP Appl No. 2005-301154 filed 20051017); Japanese Publication No.2006008718, published 20060112 (JP Appl No. 2005-301152 filed 20051017);Japanese Publication No. 2006008713, published 20060112 (JP Appl No.2005-301147 filed 20051017); Japanese Publication No. 2006008715,published 20060112 (JP Appl No. 2005-301149 filed 20051017); JapanesePublication No. 2006008714, published 20060112 (JP Appl No. 2005-301148filed 20051017); and Japanese Publication No. 2006008712, published20060112 (JP Appl No. 2005-301146 filed 20051017).

It will be appreciated that, in some embodiments of the invention,carotenoids produced by manipulated host cells as described herein areincorporated into a final product (e.g., food or feed supplement,pharmaceutical, cosmetic, dye-containing item, etc.) in the context ofthe host cell. For example, host cells may be lyophilized, freeze dried,frozen or otherwise inactivated, and then whole cells may beincorporated into or used as the final product. The host cell may alsobe processed prior to incorporation in the product to increasebioavailability (e.g., via lysis). Alternatively or additionally, afinal product may incorporate only a portion of the host cell (e.g.,fractionated by size, solubility), separated from the whole. Forexample, in some embodiments of the invention, lipid droplets areisolated from the host cells and are incorporated into or used as thefinal product. In other embodiments, the carotenoids themselves, orindividual carotenoid compounds are isolated and reformulated into thefinal product.

As stated above, fatty acid and glucoside esters are the predominantcarotenoid esters found in nature, whereas additional esters (e.g. withorganic acids or inorganic phosphate) can be synthesized to generateuseful product forms. For delivery, carotenoid esters can also beformulated as salts of the ester form. See, e.g., US Publication No.20050096477.

The amount of carotenoid incorporated into a given product may varydramatically depending on the product, and the particular carotenoid(s)involved. Amounts may range, for example, from less than 0.01% by weightof the product, to more than 1%, 10%, 20%, 30% or more; in some casesthe carotenoid may comprise 100% of the product.

In some embodiments of the invention, one or more produced carotenoidsis incorporated into a component of food or feed (e.g., a foodsupplement). Types of food products into which carotenoids can beincorporated according to the present invention are not particularlylimited, and include beverages such as teas, juices, and liquors;confections such as jellies and biscuits; fat-containing foods andbeverages such as dairy products; processed food products such as riceand soft rice (or porridge); infant formulas; or the like. In someembodiments of this aspect of the invention, it may be useful toincorporate the carotenoids within bodies of edible lipids as it mayfacilitate incorporation into certain fat-containing food products.

Examples of feedstuffs into which carotenoids produced in accordancewith the present invention may be incorporated include, for instance,pet foods such as cat foods, dog foods and the like, feeds for aquariumfish, cultured fish or crustaceans, etc., feed for farm-raised animals(including livestock and further including fish or crustaceans raised inaquaculture). Food or feed material into which the carotenoid(s)produced in accordance with the present invention is incorporated ispreferably palatable to the organism which is the intended recipient.This food or feed material may have any physical properties currentlyknown for a food material (e.g., solid, liquid, soft).

In some embodiments of the invention, one or more produced carotenoidsis incorporated into a cosmetic product. Examples of such cosmeticsinclude, for instance, skin cosmetics (e.g., lotions, emulsions, creamsand the like), lipsticks, anti-sunburn cosmetics, makeup cosmetics,fragrances, products for daily use (e.g., toothpastes, mouthwashes, badbreath preventive agents, solid soaps, liquid soaps, shampoos,conditioners), etc.

In some embodiments, one or more produced carotenoids is incorporatedinto a pharmaceutical. Examples of such pharmaceuticals include, forinstance, various types of tablets, capsules, drinkable agents, troches,gargles, etc. In some embodiments, the pharmaceutical is suitable fortopical application. Dosage forms are not particularly limited, andinclude capsules, oils, granula, granula subtilae, pulveres, tabellae,pilulae, trochisci, or the like. Oils and oil-filled capsules mayprovide additional advantages both because of their lack of ingredientdecomposition during manufacturing, and because inventivecarotenoid-containing lipid droplets may be readily incorporated intooil-based formulations.

Pharmaceuticals according to the present invention may be preparedaccording to techniques established in the art including, for example,the common procedure as described in the United States Pharmacopoeia,for example.

Carotenoids produced according to the present invention may beincorporated into any pigment-containing product including, for example,fabric, paint, etc. They may also be incorporated into a product whichis an environmental indicator, or an instrument such as a biosensor foruse as a detection agent.

EXEMPLIFICATION

Table 26 below describes certain Yarrowia lipolytica strains used in thefollowing exemplification:

TABLE 26 Yarrowia lipolytica strains. NRRL Y-1095 Wild type diploidATCC76861 MATB ura2-21 lyc1-5 LYS1-5B ATCC76982 MATB ade 1 leu2-35lyc1-5 xpr2 ATCC201249 MATA ura3-302 leu2-270 lys8-11 PEX17-HA MF346MATA ura2-21 ATCC76861 × ATCC201249 MF350 MATB ura2-21 leu2-35 ade lATCC76982 × MF346

(The genotypes at LYC1, LYS1, XPR2, and PEX17 were not determined incrosses nor verified for ATCC strains.)

All basic molecular biology and DNA manipulation procedures describedherein are generally performed according to Sambrook et al. or Ausubelet al. (Sambrook J, Fritsch E F, Maniatis T (eds). 1989. MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: NewYork; Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, SmithJ A, Struhl K (eds). 1998. Current Protocols in Molecular Biology.Wiley: New York).

Example 1 Production of Plasmids for Carotenoid Strain Construction

Plasmids were generated for construction of carotenoid producingstrains. The following subparts describe production of plasmids encodingcarotenogenic polypeptides. Plasmids used in these studies and detailsof their construction are described in Table 27. Additional plasmidconstruction details and descriptions of their use are found in the textof the relevant subsection. All PCR amplifications used NRRL Y-1095genomic DNA as template unless otherwise specified. The URA5 genedescribed below is allelic with the ura2-21 auxotrophy above. The GPD1and TEF1 promoters are from Y. lipolytica as is the XPR2 terminator.

GGS1 is the gene encoding the Y. lipolytica gene encodinggeranylgeranylpyrophosphate synthase. The nucleic acid coding sequence,and encoded Ggs1 protein of pMB4591 and pMB4683 are as follows:

               atggattataacagcgcggatttcaaggagatatggggcaaggccgccgacaccgcgctgctgggaccgtacaactacctcgccaacaaccggggccacaacatcagagaacacttgatcgcagcgttcggagcggttatcaaggtggacaagagcgatctcgagaccatttcgcacatcaccaagattttgcataactcgtcgctgcttgttgatgacgtggaagacaactcgatgctccgacgaggcctgccggcagcccattgtctgtttggagtcccccaaaccatcaactccgccaactacatgtactttgtggctctgcaggaggtgctcaagctcaagtcttatgatgccgtctccattttcaccgaggaaatgatcaacttgcatagaggtcagggtatggatctctactggagagaaacactcacttgcccctcggaagacgagtatctggagatggtggtgcacaagaccggtggactgtttcggctggctctgagacttatgctgtcggtggcatcgaaacaggaggaccatgaaaagatcaactttgatctcacacaccttaccgacacactgggagtcatttaccagattctggatgattacctcaacctgcagtccacggaattgaccgagaacaagggattctgcgaagatatcagcgaaggaaagttttcgtttccgctgattcacagcatacgcaccaacccggataaccacgagattctcaacattctcaaacagcgaacaagcgacgcttcactcaaaaagtacgccgtggactacatgagaacagaaaccaagagtttcgactactgcctcaagaggatacaggccatgtcactcaaggcaagttcgtacattgatgatctagcagcagctggccacgatgtctccaagctacgagccattttgcattattttgtgtccacctctgactgtgaggagagaaagtactttgaggatgcgcagtga               mdynsadfkeiwgkaadtallgpynylannrghnirehliaafgavikvdksdletishitkilhnssllvddvednsmlrrglpaahclfgvpqtinsanymyfvalqevlklksydavsifteeminlhrgqgmdlywretltcpsedeylemvvhktgglfrlalrlmlsvaskqedhekinfdlthltdtlgviyqilddylnlqsteltenkgfcedisegkfsfplihsirtnpdnheilnilkqrtsdaslkkyavdymrtetksfdyclkriaamslkassyiddlaaaghdvsklrailhyfvstsdceerkyfedaq

TABLE 27 Plasmids Plasmid Backbone Insert Oligos or source pMB4529PCR2.1 3.4 kb ADE1 PCR product MO4475 & MO4476 pMB4534 PCR2.1 2.1 kbLEU2 PCR product MO4477 & MO4478 pMB4535 PCR2.1 1.2 kb URA5 PCR productMO4471 & MO4472 pMB4589 pMB4535 (KpnI + SpeI) 1.2 kb GPD1 promoter(KpnI + MO4568 & NotI); 0.14 kb XPR2 MO4591; MO4566 terminator (NotI +SpeI) & MO4593 pMB4590 pMB4535 (KpnI + SpeI) 0.4 kb TEF1 promoter(KpnI + MO4571 & NotI); 0.14 kb XPR2 MO4592; MO4566 terminator (NotI +SpeI) & MO4593 pMB4591 pMB4590 (NheI + MluI) 1.0 kb GGS1 ORF (XbaI +MO4534 & MO4544 MluI) pMB4597 pMB4534 (Acc65I + SpeI) GPD1 promoter &XPR2 From pMB4589 terminator (Acc65I + SpeI) pMB4603 pMB4597 (RsrII +MluI) Residual backbone From pMB4590 & TEF1 promoter (RsrII + MluI)pMB4616 pMB4529 (RsrII + SpeI) Residual backbone From pMB4589 & GPD1promoter & XPR2 terminator (RsrII + SpeI) pMB4629 pMB4616 (RsrII + MluI)Residual backbone From pMB4590 & TEF1 promoter (RsrII + MluI) pMB4631pMB4603 (KpnI + NheI) 1.2 kb GPD1 promoter (KpnI + MO4568 & MO4659NheI); pMB4628 pMB4603 Carp See 1A pMB4637 pMB4629 (NheI + MluI) 1.5 kbhmg1^(trunc) ORF (XbaI + See 1D MluI) pMB4638 pMB4629 carB(i⁻) See 1BpMB4660 pMB4638 (+URA3) carB(i⁻) See 1C pMB4662 pMB4631 (SpeI + XhoI)1.8 kb URA3 fragment (SpeI + MO4684 & MO4685 BsaI See 1C pMB4683 pMB4662(Acc65I + MluI) 1.4 kb tef1p-GGS1 fragment From pMB4591 (Acc65I + MluI)pMB4692 pMB4662 (Acc65I + MluI) 0.4 kb TEF1 promoter See 1E (Acc65I +NheI); 0.55 kb crtZ ORF (XbaI + MluI) pMB4698 pMB4629 (NheI + MluI) 0.9kb crtW ORF (XbaI + See 1F MluI) pMB4599 pBluescriptSKII- 1.9 kb carRPgene MO4525 & MO4541 (EcoRV) pMB4606 pBluescriptSKII- 1.9 kb carB geneMO4530 & (EcoRV) MO4542 pMB4613 pMB4599 (Acc65I + carRP(i⁻) See textPpuMI) pMB4619 pBluescriptSKII- carB(i⁻) See text (BamHI + Acc65I))

Certain oligonucleotides referred to in Table 27 above are as follows:

MO4471 5′-CTGGGTGACCTGGAAGCCTT MO4472 5′-AAGATCAATCCGTAGAAGTTCAG MO44755′-AAGCGATTACAATCTTCCTTTGG MO4476 5′-CCAGTCCATCAACTCAGTCTCA MO44775′-GCATTGCTTATTACGAAGACTAC MO4478 5′-CCACTGTCCTCCACTACAAACAC MO45345′-CACAAACGCGTTCACTGCGCATCCTCAAAGT MO45445′-CACAATCTAGACACAAATGGATTATAACAGCGCGGAT MO45665′-CACAAACTAGTTTGCCACCTACAAGCCAGAT MO45685′-CACAAGGTACCAATGTGAAAGTGCGCGTGAT MO45715′-CACAAGGTACCAGAGACCGGGTTGGCGG MO45915′-CACAAGCGGCCGCGCTAGCATGGGGATCGATCTCTTATAT MO45925′-CACAAGCGGCCGCGCTAGCGAATGATTCTTATACTCAGAAG MO45935′-CACAAGCGGCCGCACGCGTGCAATTAACAGATAGTTTGCC MO46595′-CACAAGCTAGCTGGGGATGCGATCTCTTATATC

1A: Production of pMB4628 (tef1p-carRP LEU2) encoding phytoenesynthase/lycopene cyclase: Intron-containing carRP was amplified from M.circinelloides (ATCC 90680) genomic DNA using M04525 and M04541:

MO4525 5′-CACAAACGCGTTTAAATGGTATTTAGATTTCTCATT MO45415′-CACAATCTAGACACAAATGCTGCTCACCTACATGGAand the resulting 1.9 kb fragment was phosphorylated with T4polynucleotide kinase. The resulting fragment was blunt-end ligated intopBluescriptSKII— cleaved with EcoRV, yielding pMB4599. The 1.9 kbXbaI-MluI fragment from pMB4599 was inserted into NheI- and MluI-cleavedpMB4603, yielding pMB4628. The intron containing nucleic acid codingsequence, and encoded CarRP protein of pMB4628 are as follows:

               atgctgctcacctacatggaagtccacctctactacacgctgcctgtgctgggcgtcctgtcctggctgtcgcggccgtactacacagccaccgatgcgctcaaattcaaatttctgacactggttgccttcacgaccgcctccgcctgggacaactacattgtctaccacaaggcgtggtcctactgccccacctgcgtcaccgctgtcattggctacgtgcccttggaggagtacatgttcttcatcatcatgactctgttgaccgtggcattcaccaatctggtgatgcgctggcacctgcacagcttctttatcaggcctgaaacgcccgtcatgcagtccgtcctggtccgtcttgtccccataacagccttattaatcactgcatacaaggcttgggtaagcaaacaaacaaatgatgtgccgcatcgcattttaatattaaccattgcatacacagcatttggcggtccctggaaagccactgttctacggatcatgcattttgtggtacgcctgtccggttttggccttattgtggtttggtgctggcgagtacatgatgcgtcgtccgctggcggtgctcgtctccattgcgctgcccacgctgtttctctgctgggtcgatgtcgtcgctattggcgccggcacatgggacatttcgctggccacaagcaccggcaagttcgtcgtgccccacctgcccgtggaggaattcatgttctttgcgctaattaataccgttttggtatttggtacgtgtgcgatcgatcgcacgatggcgatcctccacctgttcaaaaacaagagtccttatcagcgcccataccagcacagcaagtcgttcctccaccagatcctcgagatgacctgggccttctgtttacccgaccaagtgctgcattcagacacattccacgacctgtccgtcagctgggacatcctgcgcaaggcctccaagtccttttacacggcctctgctgtctttcccggcgacgtgcgccaagagctcggtgtgctatacgccttttgcagagccacggacgatctctgcgacaacgagcaggtccctgtgcagacgcgaaaggagcagctgatactgacacatcagttcgtcagcgatctgtttggccaaaagacaagcgcgccgactgccattgactgggacttttacaacgaccaactgcctgcctcgtgcatctctgccttcaagtcgttcacccgtttgcgccatgtgctggaagctggagccatcaaggaactgctcgacgggtacaagtgggatttggagcgtcgctccatcagggatcaggaggatctcagatattactcagcttgtgtcgccagcagtgttggtgaaatgtgcactcgcatcatactggcccacgccgacaagcccgcctcccgccagcaaacacagtggatcattcagcgtgcgcgtgaaatgggtctggtactccaatatacaaacattgcaagagacattgtcaccgacagcgaggaactgggcagatgctacctgcctcaggattggcttaccgagaaggaggtggcgctgattcaaggcggccttgcccgagaaattggcgaggagcgattgctctcactgtcgcatcgcctcatctaccaggcagacgagctcatggtggttgccaacaagggcatcgacaagctgcccagccattgtcaaggcggcgtgcgtgcggcctgcaacgtctatgcttccattggcaccaagctcaagtcttacaagcaccactatcccagcagagcacatgtcggcaattcgaaacgagtggaaattgctcttcttagcgtatacaacctttacaccgcgccaattgcgactagtagtaccacacattgcagacagggaaaaatg agaaatctaaataccatttaa               mlltymevhlyytlpvlgvlswlsrpyytatdalkfkfltlvafttasawdnyivyhkawsycptcytavigyvpleeymffiimtlltvaftnlvmrwhlhsffirpetpvmqsvlvrlvpitallitaykawhlavpgkplfygscilwyacpvlallwfgageymmrrplavlvsialptlflcwvdvvaigagtwdislatstgkfvvphlpveefmffalintvlvfgtcaidrtmailhlfknkspyqrpyqhsksflhqilemtwafclpdqvlhsdtfhdlsvswdilrkasksfytasavfpgdvrqelgvlyafcratddlcdneqvpvqtrkeqlilthqfvsdlfgqktsaptaidwdfyndqlpascisafksftrlrhvleagaikelldgykwdlerrsirdqedlryysacvassvgemctriilahadkpasrqqtqwiiqraremglvlqytniardivtdseelgrcylpqdwltekevaliqgglareigeerllslshrliyqadelmvvankgidklpshcqggvraacnvyasigtklksykhhypsrahvgnskrveiallsvynlytapiatsstthcrqgkmrnlnti

Alternatively, pMB4599 was also used as a template for PCR amplificationusing MO4318, MO4643, MO4644, and MO4639 and

MO4318 5′-GTAAAACGACGGCCAGT MO46435′-CACACGGTCTCATGCCAAGCCTTGTATGCAGTGATTAA MO4639 5′-CCACTGTGTTTGCTGGCGGMO4644 5′-CACACGGTCTCTGGCATTTGGCGGTCCCTGGAAAproducing fragments of 0.5 and 0.95 kb, that were subsequently cleavedwith Acc65I and BsaI, and BsaI and PpuMI, respectively. These fragmentswere ligated to pMB4599 that had been digested with Acc65I and PpuMI,yielding pMB4613, harboring intronless carRP. The 1.85 kb XbaI-MluIfragment from pMB4613 can be inserted into NheI- and MluI-cleavedpMB4603 to yield pCarRPdeII.

1B: Production of pMB4638 (tef1 p-carB ADE1), encoding phytoenedehydrogenase: Intron-containing carB was amplified from M.circinelloides (ATCC 90680) genomic DNA using MO4530 and MO4542:

MO4530 5′-CACAAACGCGTTTAAATGACATTAGAGTTATGAAC MO45425′-CACAATCTAGACACAAATGTCCAAGAAACACATTGTCand the resulting 1.9 kb fragment was phosphorylated with T4polynucleotide kinase and blunt-end ligated into pBS-SKII- cleaved withEcoRV, yielding pMB4606. pMB4606 was then used as a template for PCRamplification using MO4318 and MO4648, and MO4646 and MO4647, and MO4343and MO4645:

MO4318 5′-GTAAAACGACGGCCAGT MO4648 5′-CACAAGGTCTCAAGCACGCATCCCGGAACTGMO4646 5′-CACACGGTCTCAGGCATGTCGCCCTACGATGC MO46475′-CACACGGTCTCATGCTTGCACCCACAAAGAATAGG MO4343 5′-CAGGAAACAGCTATGACMO4645 5′-CACACGGTCTCTTGCCCATATACATGGTCTGAAACGproducing fragments of 0.4 and 0.85 and 0.7 kb, that were subsequentlycleaved with Acc65I and BsaI, and BsaI, and BsaI and BamHI,respectively. These fragments were ligated to pBS-SKII- that had beencut with Acc65I and BamHI, yielding pMB4619, harboring intronless carB.The 1.75 kb XbaI-MluI fragment from pMB4619 was inserted into NheI- andMluI-cleaved pMB4629, yielding pMB4638. The resulting nucleic acidcoding sequence and encoded CarB protein of pMB4638 are as follows:

               atgtccaagaaacacattgtcattatcggtgctggcgtgggtggcacggctacagctgctcgtttggcccgcgaaggcttcaaggtcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatccatcaccagggccatcgctttgatcagggcccgtcgctctacctgatgcccaagtactttgaggacgcctttgccgatctggacgagcgcattcaagaccacctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgacggtgagtcgatccagctgtcgtctgacttgacacgcatgaaggctgaattggaccgcgtggagggcccccttggttttggccgattcctggatttcatgaaagagacacacatccactacgaaagcggcaccctgattgcgctcaagaagaatttcgaatccatctgggacctgattcgcatcaagtacgctccagagatctttcgcttgcacctgtttggcaagatctacgaccgcgcttccaagtacttcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatgggcatgtcgccctacgatgcgcctgctgtctacagcctgttgcagtacaccgagttcgctgaaggcatctggtatccccgtggcggcttcaacatggtggttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatctacaatgcgcctgttgccaagattaacaccgatgatgccaccaaacaagtgacaggtgtaaccttggaaaatggccacatcatcgatgccgatgcggttgtgtgtaacgcagatctggtctatgcttatcacaatctgttgcctccctgccgatggacgcaaaacacactggcttccaagaaattgacgtcttcttccatttcctctactggtccatgtccaccaaggtgcctcaattggacgtgcacaacatctttttggccgaggcttatcaggagagctttgacgaaatcttcaaggactttggcctgccttctgaagcctccttctacgtcaatgtgccctctcgcatcgatccttctgctgctcccgacggcaaggactctgtcattgtcttggtgcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaactacccggccatggtggacaaggcacgcaagatggtgctggctgtgattgagcgtcgtctgggcatgtcgaatttcgccgacttgattgagcatgagcaagtcaatgatcccgctgtatggcagagcaagttcaatctgtggagaggctcaattctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccagcacaaaggattctaccggtcgttatgataacctattctttgtgggtgcaagcacgcatcccggaactggtgttcccattgtccttgcaggaagcaagctcacctctgaccaagttgtcaagagctttggaaagacgcccaagccaagaaagatcgagatggagaacacgcaagcacctttggaggagcctgatgctgaatcgacattccctgtgtggttctggttgcgcgctgccttttgggtcatgtttatgttcttttacttcttccctcaatccaatggccaaacgcccgcatcttttatcaataatttgttacctgaagtattccgcgttcataactctaatgtcat ttaa               mskkhiviigagvggtataarlaregfkvtvvekndfgggrcslihhqghrfdqgpslylmpkyfedafadlderiqdhlellrcdnnykvhfddgesiqlssdltrmkaeldrvegplgfgrfldfmkethihyesgtfialkknfesiwdlirikyapeifrlhlfgkiydraskyfktkkmrmaftfqtmymgmspydapavysllqytefaegiwyprggfnmvvqkleaiakqkydaefiynapvakintddatkqvtgvtlenghiidadavvcnadlvyayhnllppcrwtqntlaskkltsssisfywsmstkvpqldvhniflaeayqesfdeifkdfglpseasfyvnvpsridpsaapdgkdsvivlvpighmksktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavwqskfnlwrgsilglshdvlqvlwfrpstkdstgrydnlffvgasthpgtgvpivlagskltsdqvvksfgktpkprkiementqapleepdaestfpvwfwlraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi

1C. Production of pMB4660 (tef1 p-carB URA3) encoding phytoenedehydrogenase: The 4.3 kb XhoI-NotI fragment and the 1.8 kb NotI-SpeIfragment from pMB4638 were ligated to the 1.9 kb BsaI- and SpeI-cleavedURA3 gene generated by PCR amplification of Y lipolytica genomic DNAusing MO4684 and MO4685 to create pMB4660:

MO4684 5′-CATTCACTAGTGGTGTGTTCTGTGGAGCATTC MO46855′-CACACGGTCTCATCGAGGTGTAGTGGTAGTGCAGTGThe resulting nucleic acid coding sequence and encoded CarB(i) proteinof pMB4660 are as follows:

               atgtccaagaaacacattgtcattatcggtgctggcgtgggtggcacggctacagctgctcgtttggcccgcgaaggcttcaaggtcactgtggtggagaaaaacgactttggtggcggccgctgctccttgatccatcaccagggccatcgctttgatcagggcccgtcgctctacctgatgcccaagtactttgaggacgcctttgccgatctggacgagcgcattcaagaccacctggagctgctgcgatgcgacaacaactacaaggtgcactttgacgacggtgagtcgatccagctgtcgtctgacttgacacgcatgaaggctgaattggaccgcgtggagggcccccttggttttggccgattcctggatttcatgaaagagacacacatccactacgaaagcggcaccctgattgcgctcaagaagaatttcgaatccatctgggacctgattcgcatcaagtacgctccagagatctttcgcttgcacctgtttggcaagatctacgaccgcgcttccaagtacttcaagaccaagaagatgcgcatggcattcacgtttcagaccatgtatatgggcatgtcgccctacgatgcgcctgctgtctacagcctgttgcagtacaccgagttcgctgaaggcatctggtatccccgtggcggcttcaacatggtggttcagaagctagaggcgattgcaaagcaaaagtacgatgccgagtttatctacaatgcgcctgttgccaagattaacaccgatgatgccaccaaacaagtgacaggtgtaaccttggaaaatggccacatcatcgatgccgatgcggttgtgtgtaacgcagatctggtctatgcttatcacaatctgttgcctccctgccgatggacgcaaaacacactggcttccaagaaattgacgtcttcttccatttccttctactggtccatgtccaccaaggtgcctcaattggacgtgcacaacatctttttggccgaggcttatcaggagagctttgacgaaatcttcaaggactttggcctgccttctgaagcctccttctacgtcaatgtgccctctcgcatcgatccttctgctgctcccgacggcaaggactctgtcattgtcttggtgcctattggtcatatgaagagcaagacgggcgatgcttccaccgagaactacccggccatggtggacaaggcacgcaagatggtgctggctgtgattgagcgtcgtctgggcatgtcgaatttcgccgacttgattgagcatgagcaagtcaatgatcccgctgtatggcagagcaagttcaatctgtggagaggctcaattctgggtttgtctcatgatgtgcttcaggtgctgtggttccgtcccagcacaaaggattctaccggtcgttatgataacctattctttgtgggtgcaagcacgcatcccggaactggtgttcccattgtccttgcaggaagcaagctcacctctgaccaagttgtcaagagctttggaaagacgcccaagccaagaaagatcgagatggagaacacgcaagcacctttggaggagcctgatgctgaatcgacattccctgtgtggttctggttgcgcgctgccttttgggtcatgtttatgttcttttacttcttccctcaatccaatggccaaacgcccgcatcttttatcaataatttgttacctgaagtattccgcgttcataactctaatgtca tttaa               mskkhiviigagvggtataarlaregfkvtvvekndfgggrcslihhqghrfdqgpslylmpkyfedafadlderiqdhlellrcdnnykvhfddgesiqlssdltrmkaeldrvegplgfgrfldfmkethihyesgtlialkknfesiwdlirikyapeifrlhlfgkiydraskyfktkkmrmaftfqtmymgmspydapavysllqytefaegiwyprggfnmvvqkleaiakqkydaefiynapvakintddatkqvtgvtlenghiidadavvcnadlvyayhnllppcrwtqntlaskkltsssisfywsmstkvpqldvhniflaeayqesfdeifkdfglpseasfyvnvpsridpsaapdgkdsvivlvpighmksktgdastenypamvdkarkmvlavierrlgmsnfadlieheqvndpavwqskfnlwrgsilglshdvlqvlwfrpstkdstgrydnlffvgasthpgtgvpivlagskltsdqvvksfgktpkprkiementqapleepdaestfpvwfwlraafwvmfmffyffpqsngqtpasfinnllpevfrvhnsnvi

1D. Production of pMB4637 and pTef-HMG encoding a truncated HMG1. Forproduction of a truncated variant of the HMG-CoA reductase gene, whichalso encodes a 77 amino acid leader sequence derived from S. cerevisiae,the following oligonucleotides are synthesized:

PRIMER O 5′-TTCTAGACACAAAAATGGCTGCAGACCAATTGGTGA PRIMER P5′-CATTAATTCTTCTAAAGGACGTATTTTCTTATC PRIMER Q 5′-GTTCTCTGGACGACCTAGAGGMO4658 5′-CACACACGCGTACACCTATGACCGTATGCAAATPrimers O and P are used to amplify a 0.23 kb fragment encoding Met-Alafollowed by residues 530 to 604 of the Hmg1 protein of S. cerevisiae,using genomic DNA as template. Primers Q and MO4658 are used to amplifya 1.4 kb fragment encoding the C-terminal 448 residues of the Hmg1protein of Y. lipolytica, using genomic DNA as template. These fragmentsare ligated to the appropriate cloning vector, and the resultantplasmids, designated pOP and pQMO4658, are verified by sequencing. TheOP fragment is liberated with XbaI and AseI, and the QMO4658 fragment isliberated with MaeI and MluI. These fragments are then ligated to theADE1 TEF1p expression vector pMB4629 cut with XbaI and MluI to producepTefHMG.

Alternatively, the native HMG1 gene from Y. lipolytica may be modifiedwithout S. cerevisiae sequences as described in the table above usingprimers MO4658 (described above) and MO4657, to create pMB4637:

MO4657 5′-CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGGThe resulting nucleic acid coding sequence and encoded Hmg1^(trune)protein of pMB4637 are as follows:

               atgacccagtctgtgaaggtggttgagaagcacgttcctatcgtcattgagaagcccagcgagaaggaggaggacacctcttctgaagactccattgagctgactgtcggaaagcagcccaagcccgtgaccgagacccgttctctggacgacctagaggctatcatgaaggcaggtaagaccaagcttctggaggaccacgaggttgtcaagctctctctcgagggcaagcttcctttgtatgctcttgagaagcagcttggtgacaacacccgagctgttggcatccgacgatctatcatctcccagcagtctaataccaagactttagagacctcaaagcttccttacctgcactacgactacgaccgtgtttttggagcctgttgcgagaacgttattggttacatgcctctccccgttggtgttgctggccccatgaacattgatggcaagaactaccacattcctatggccaccactgagggttgtcttgttgcctcaaccatgcgaggttgcaaggccatcaacgccggtggcggtgttaccactgtgcttactcaggacggtatgacacgaggtccttgtgtttccttcccctctctcaagcgggctggagccgctaagatctggcttgattccgaggagggtctcaagtccatgcgaaaggccttcaactccacctctcgatttgctcgtctccagtctcttcactctacccttgctggtaacctgctgtttattcgattccgaaccaccactggtgatgccatgggcatgaacatgatctccaagggcgtcgaacactctctggccgtcatggtcaaggagtacggcttccctgatatggacattgtgtctgtctcgggtaactactgcactgacaagaagcccgcagcgatcaactggatcgaaggccgaggcaagagtgttgttgccgaagccaccatccctgctcacattgtcaagtctgttctcaaaagtgaggttgacgctcttgttgagctcaacatcagcaagaatctgatcggtagtgccatggctggctctgtgggaggtttcaatgcacacgccgcaaacctggtgaccgccatctaccttgccactggccaggatcctgctcagaatgtcgagtcttccaactgcatcacgctgatgagcaacgtcgacggtaacctgctcatctccgtttccatgccttctatcgaggtcggtaccattggtggaggtactattttggagccccagggggctatgctggagatgcttggcgtgcgaggtcctcacatcgagacccccggtgccaacgcccaacagcttgctcgcatcattgcttctggagttcttgcagcggagctttcgctgtgttctgctcttgctgccggccatcttgtgcaaagtcatatgacccacaaccggtcccaggctcctactccggccaagcagtctcaggccgatctgcagcgtctacaaaacggttcgaat atttgcatacggtcatag               mtqsvkvvekhvpiviekpsekeedtssedsieltvgkqpkpvtetrslddleaimkagktklledhevvklslegklplyalekqlgdntravgirrsiisqqsntktletsklpylhydydrvfgaccenvigymplpvgvagpmnidgknyhipmattegclvastmrgckainagggvttvltqdgmtrgpcvsfpslkragaakiwldseeglksmrkafnstsrfarlqs1hstlagnllfirfrtttgdamgmnmiskgvehslavmvkeygfpdmdivsysgnyctdkkpaainwiegrgksvvaeatipahivksvlksevdalvelnisknligsamagsvggfnahaanlvtaiylatgqdpaqnvessncitlmsnvdgnllisvsmpsievgtigggtilepqgamlemlgvrgphietpganaqq1ariiasgvlaaelslcsalaaghlvqshmthnrsqaptpakqsqa dlqrlqngsnicirs

1E. Production of pMB4692 (URA3 tef1p-crtZ) encoding carotenehydroxylase. The following carotene hydroxylase (CrtZ) ORF sequence wassynthesized; based on protein sequence of Novosphingobiumaromaticivorans, using Y. lipolytica codon bias:

5′- ttctagacacaaaa atgggtggagccatgcagaccctcgctgctatcctgatcgtcctcggtacagtgctcgctatggagtttgtcgcttggtcttctcataagtatatcatgcatggcttcggatggggatggcatagagaccatcacgagccccatgagggatttcttgagaagaatgacttatacgccatcgttggcgctgccctctcgatactcatgtttgccctcggctctcccatgatcatgggcgctgacgcctggtggcccggaacctggatcggactcggtgtcctcttctatggtgtcatctataccctcgtgcaccacggtctggtgcaccaacgatggtttagatgggtgcctaaacgaggttacgccaaacgactcgtgcaggcccataagctgcaccacgccaccattggcaaggaaggaggcgtctcattcggtttcgtgttcgcccgagatcccgccgttctgaagcaggagcttcgagctcaacgagaagcaggtatcgccgtgctgcgagaggctgtggacggc tagacgcg t

This sequence was cleaved using XbaI and MluI and ligated, along with anAcc651-NheI TEF1 promoter fragment from pMB4629, to pMB4662 cut withAcc65I and MluI to produce pMB4692. The nucleic acid coding sequence isdepicted in bold underline above. The resulting encoded crtZ protein ofpMB4692 is as follows:

               mggamqtlaailivlgtvlamefvawsshkyimhgfgwgwhrdhhephegflekndlyaivgaalsilmfalgspmimgadawwpgtwiglgvlfygviytlvhdglvhqrwfrwvpkrgyakrlvqahklhhatigkeggvsfgfvfardpavlkqelraqreagiavlreavdg

1F. Production of pMB4698 (ADE1 tef1p-crtW), encoding carotene ketolase.The following carotene ketolase (CrtW) ORF sequence was synthesized,based on protein sequence of an environmental sequence isolated from theSargasso Sea (Genbank accession AACY01034193.1):

5′- ttctagacacaaaaa tgactcgatctatttcctggccttccacctactggcacctccagccctcctgttcttcttgggtcgcaaacgaattctctcctcaagcccgaaaaggtctcgtcctcgctggtctcattggttccgcttggctgcttactctcggacttggcttttcccttcccctccatcaaacgagctggcttctcatcggttgtctcgttctccttagatctttcctgcacaccggactttttatcgttgcccatgacgctatgcacgcttctcttgttcctgaccaccctggccttaaccgttggattggacgtgtctgtcttctcatgtatgctggactctcctacaaaagatgctgccgaaatcaccgtcgacaccaccaagcccctgaaacagttgaagaccctgactaccaacgatgcactaacaacaatatcctcgactggtacgttcactttatgggaaattacctcggatggcaacaattgcttaatctctcttgcgtttggctcgctctcaccttccgtgtttctgactactctgctcaattcttccacctgctccttttctctgtccttcctctcatcgtctcctcctgtcaactcttcctcgtgggaacctggctgccacaccgacgaggcgctactactcgacccggcgttaccactcgatccctgaacttccaccctgctctttccttcgctgcttgctaccacttcggttaccaccgtgaacaccatgaatctccctctactccttggttccaacttcctaaactccgagaaggttct ctcatctaa acgcgtThis sequence was cleaved using XbaI and MluI and ligated to pMB4629 cutwith NheI and MluI to produce pMB4698. The nucleic acid coding sequenceis depicted in bold underline above. The resulting encoded crtW proteinof pMB4698 is as follows:

mtrsiswpstywhlqpscsswvanefspqarkglvlagligsawlltlglgfslplhqtswlligclvllrsflhtglfivahdamhaslvpdhpglnrwigrvcllmyaglsykrccmhnhhqapetvedpdyqrannnildwyvhfmgnylgwqqllnlscvwlaltfrvsdysaqffhlllfsvlplivsscqlflvgtwlphrrgattrpgvttrslnfhpalsfaacyhfgyhrehhespstpwfqlpklregsli

Example 2 Engineering Yarrowia lipolytica for Increased CarotenoidProduction

2A. Production of Y. lipolytica expressing geranylgeranylpyrophosphatesynthase and phytoene dehydrogenase: MF350 (MATB ura2-21 leu2-35 ade1)was transformed with pMB4591 (tef1p-GGSI) that had been cleaved upstreamof URA5 with SspI; a Ura⁺ transformant carrying the plasmid at the ura2locus was identified and named MF364. It was subsequently transformedwith pMB4638 (tef1p-carB) that had been cleaved at ADE1 with SspI and aprototrophic transformant was chosen that harbored the plasmid at theade1 locus. This strain was named MF502.

2B. Production of Y. lipolytica expressing geranylgeranylpyrophosphatesynthase, phytoene dehydrogenase and phytoene synthase/lycopene cyclaseMF502 was transformed with pMB4628 (tef1p-carRP) that had been treatedwith SspI. Nine prototrophic colonies were chosen that were uncolored,orange, or very orange on the transformation plate (YNB agar with 1%glucose and 0.1% glutamate [YNBglut]) after two to three days of growth.Two, MF597 and MF600 (the very orange ones), produced greater than 4 mgcarotene per g dry cell weight (DCW) after four days of growth in YPD at30° C. Southern analysis reveals a different single KpnI-HindIII band ingenomic DNA from MF597 and MF600, neither of which suggested thathomologous integration occurred at leu2-270.

2C. Production of Y lipolytica expressing phytoene synthase/lycopenecyclase and phytoene dehydrogenase: ATCC201249 (MATA ura3-302 leu2-270lys8-11) was transformed with SspI-cleaved pMB4628. Hundreds of Leu⁺colonies were pooled, re-grown, and transformed with pMB4660(tef1p-carB) that had been cleaved upstream of URA3 with SalI. Onecolony that was noticeably yellow after 5 days at 30° C. on YNBglut plus0.6 mM lysine was selected, named MF447, and found to produce 0.2 mgcarotene per gram dry cell weight after 4 days of growth in YPD.

MF447 was challenged with 1 g/L 5-fluoroorotic acid and Ura⁻ segregantsselected. Surprisingly, they were all found to retain the identicalyellow appearance of their parent, implying that the loss of afunctional URA3 gene did not coincide with the loss of a functional CarBenzyme. Southern analysis demonstrates that two fragments from aKpnI-HindIII digest of MF447 DNA contain URA3p-hybridizing sequences,only one of which also hybridizes to carB. The other is absent in MF578,the Ura3⁻ segregant chosen for further manipulation. Plasmid rescue andanalysis of the DNA sequence encompassing the carRP intron in strainsMF447, MF597 (example 2c), and MF600 (example 2c) revealed that exons 1and 2 were contiguous and were each separated by an intron sequence thatlacked the original internal SspI site (present in pMB4628).

2D. Production of Y. lipolytica expressing phytoene synthase/lycopenecyclase, phytoene dehydrogenase and geranylgeranylpyrophosphatesynthase: MF578 was transformed with pMB4683 (tef1p-GGS1) that had beencleaved with SalI (upstream of URA3) or with StuI (within the GGS1 ORF).Ura⁺Leu⁺ colonies in both cases appeared bright orange on YNBglut+Lysand on YPD, and several produced greater than 4 mg carotene per gram ofdry cell weight when grown as above. One, MF633, contained a single copyof the plasmid at the GGS1 locus, as inferred from Southern analysis.The others arose by non-homologous or more complex integrations.

2E. Production of Y. lipolytica expressing phytoene synthase/lycopenecyclase, phytoene dehydrogenase and geranylgeranylpyrophosphatesynthase: MF364 is crossed with MF578, and spores from the resultingdiploid are plated on YPD for two to three days at 30° C. Orange Leu⁺Ade⁻ Ura⁻ colonies are screened for the presence of tefp-carB,tefp-carRP, and tefp-GGS1 by PCR, and for high carotenoid (>4 mg/g drycell weight) production after growth in YPD liquid medium. Coloniesmeeting these criteria, as well as displaying resistance to 5-fluorooticacid, an indication that they harbor the ura3-302 allele, are chosen forfurther studies and hereafter referred to as GBRPua strains. Such astrain is selected for further analysis and modification.

Example 3 Extraction of Carotenoids from Yarrowia lipolytica Cells

Shake-flask testing of generated strains was conducted using YPD medium(1% yeast extract, 2% peptone, 2% glucose). 20 ml cultures in 125 mlflasks were grown at 30° C. Y. lipolytica cells were harvested from72-96 hour cultures, and extractions were performed to determinecarotenoid form and quantity. 1.8 ml of culture was placed into anEppendorf tube. Cells were pelleted and washed twice with 1 ml H₂O.After the second wash, the resuspended cells were transferred to apre-weighed snap-cap tube with a hole poked in the top, and the cellswere lyophilized overnight. After drying to completion, the tube wasweighed in order to calculate dry cell weight. 0.25 ml from the sameshake flask culture was placed into a 2 ml screw-cap tube for carotenoidextraction. Cells were pelleted and the supernatant was aspirated.Pelleted cells may be frozen at −80° C. and stored. An equal volume ofcubic zirconia beads was added to cell pellets, along with 1 ml ice-coldextraction solvent (a 50/50 v/v mix of hexane and ethyl acetatecontaining 0.01% butylhydroxytoluene (BHT)). The mixture was thenagitated (Mini-BeadBeater-8, BioSpec Products, Inc.) at maximum speedfor 5 minutes at 4° C. The mixture was then spun at maximum speed for 1minute, and the supernatant was collected and deposited in a cold 16 mlglass vial. The remaining cell debris was re-extracted at least threetimes, without the addition of zirconia beads; all supernatants werepooled in the 16 ml glass vial. Following extraction, the glass vial wasspun for 5 minutes at 2000 rpm at 4° C. in a Sorvall tabletopcentrifuge, and the supernatant was transferred to a new cold 16 mlglass vial. A Speed Vac was used to concentrate the supernatant (roomtemperature in dark), and the samples were stored at −20° C. or −80° C.until immediately before HPLC analysis. Prior to HPLC analysis, thesamples were resuspended in 1 ml ice-cold solvent and then transferredto a cold amber vial. Throughout the protocol, care was taken to avoidcontact with oxygen, light, heat, and acids.

Example 4 Quantification of Carotenoid Production by HPLC

For carotenoid analysis, samples were resuspended in ice-cold extractionsolvent (a 50/50 v/v mix of hexane and ethyl acetate containing 0.01%butylhydroxytoluene (BHT)). An Alliance 2795 HPLC (Waters) equipped witha Waters XBridge C18 column (3.5 μm, 2.1×50 mm) and Thermo Basic 8 guardcolumn (2.1×10 mm) was used to resolve carotenoid at 25° C.; authenticcarotenoid samples were used as standards. The mobile phases and flowrates are shown below (Solvent A=Ethyl Acetate; Solvent B=Water; SolventC=Methanol; Solvent D=Acetonitrile). The injection volume was 10 μL. Thedetector is a Waters 996 photodiode array detector. The retention timesfor lipophilic molecules include astaxanthin (1.159 min), zeaxanthin(1.335), β-apo-8′-carotenal (2.86 min), ergosterol (3.11 min), lycopene(3.69 min), β-Carotene (4.02 min), and phytoene (4.13 min). Astaxanthin,zeaxanthin, β-apo-8′-carotenal, lycopene and β-Carotene are detected at475 nm, whereas ergosterol and phytoene were detected at 286 nm.

TABLE 28 Retention Times for Lipophilic Molecules Time (min) Flow(mL/min) % A % B % C % D Curve 0.50 0.0 20.0 0.0 80.0 3.00 1.00 20.0 0.00.0 80.0 6 4.50 1.00 80.0 0.0 20.0 0.0 6 5.50 1.00 0.0 0.0 60.0 40.0 66.50 1.00 0.0 0.0 80.0 20.0 6 7.50 1.00 0.0 0.0 100.0 0.0 6 8.50 1.000.0 0.0 100.0 0.0 6 9.50 1.00 0.0 20.0 0.0 80.0 6 10.50 0.50 0.0 20.00.0 80.0 6

Example 5 Expression of a Truncated Form of HMG-CoA Reductase Results inIncreased Carotenoid Production

In order to increase carotenoid production, carbon flow through theisoprenoid pathway is enhanced by introducing a truncated variant of theHMG-CoA reductase gene.

In one approach, a truncated variant of the HMG-CoA reductase gene whichalso encodes a 77 amino acid leader sequence derived from S. cerevisiaeHmg1 is introduced into a GRPBua strain (described in Example 2E above).Plasmid pTefHMG can be cleaved with SnaBI, BbvCI, or Bsu361 to directintegration at the ade1 locus, or with BamHI to direct integration atthe HMG1 locus, or with EcoRV to promote random integration, in theGRPBua strains, restoring them to adenine prototrophy. Resulting Ade⁺transformants are screened for increased carotenoid production.

Alternatively, the native HMG1 gene from Y. lipolytica may be modifiedwithout S. cerevisiae sequences as described in Example I D above, tocreate pMB4637. This plasmid can be digested as described for pTefHMGand transformed into GRPBua strains, and resulting transformantsscreened as described for increased carotenoid production.

In still another approach, a truncated variant of the N. crassa HMG-CoAreductase gene may be utilized and introduced into Y. lipolyticastrains. In order to generate a plasmid suitable for expression of theheterologous HMG-CoA reductase, p641 P (Yeast 2001; 18 (2001): 97-113)is modified by replacing the ICL1 promoter with the GPD promoter, and bythe addition of sequences conferring resistance to phleomycin. Y.lipolytica genomic DNA is amplified with two primers.

GPDdist: 5′ CACACGGTacctgtaggttgggttgggtg GPDprox: 5′CACACGGATCCtgtttaattcaagaatgaatatagagaagagaag,and the resulting fragment (0.7 kb) is cleaved with BamHI and KpnI, andligated to BamHI- and KpnI-cleaved p641P, creating the plasmid “p641Pgpd”. The ble gene under the control of the A. nidulans GPD promoter isthen excised from pBCphleo (Silar, Fungal Genetics Newsletter 42:73) asa 3.2 kb BclI-BamHI fragment and inserted into the unique BamHI site of“p641 Pgpd”, in the orientation that preserves the BamHI site proximalto the GPD promoter, to create “p641 Pgpdble”,

N. crassa genomic DNA is amplified with two primers:

Neuhmg fwd: 5′ CACACGGATCCACATCAACAatggcatctgccacccttcccc Neuhmg rev: 5′CACACGGATCcaagtgctgacgcggaacttg,and the resulting fragment is cleaved with BamHI and inserted intoBamHI-digested “p641 Pgpdble” in the correct orientation. The resultingplasmid, “pZg”, contains sequences encoding a truncated cytosoliccatalytic domain of hydroxymethylglutaryl-CoA reductase from N. crassa(Genbank accession: XP_(—)324892) under the control of the constitutiveGPD promoter. This plasmid can be introduced into the Y. lipolyticastrain created in Example 2E above, and transformants are selected bytheir resistance to phleomycin (100 μg/ml). Resulting transformants aretested for n-carotene production, as described above.

Example 6 Introduction of Heterologous Carotene Hydroxylase and CaroteneKetolase Genes into Y. Lipolytica Strains Producing Carotenoid forProduction of Astaxanthin

For introduction of carotene hydroxylase and carotene ketolase intocarotenoid producting Y. lipolytica, pMB4692 and pMB4698, described asin Example 1E and 1F above, can be sequentially introduced into theGRPBua strain (described in Example 2E). For the introduction ofpMB4692, the plasmid may be cleaved with SalI or BsrGI to directintegration at the ura3 locus, or with XbaI to promote randomintegration, selecting for uracil prototrophy. GRPBua Ura⁺ transformantsharboring pMB4692 are screened for zeaxanthin production in YPD.Zeaxanthin-producing cells are transformed with pMB4698 (which can becleaved with PpuMI, SspI or BbvC1 to direct integration at the ade1locus, or with EcoRV to promote random integration) and prototrophiccolonies are screened for astaxanthin production.

Alternatively, the order of plasmid transformation may be reversedwherein pMB4698 is transformed first and transformants are selected foradenine prototrophy. GRPBua Ade⁺ transformants harboring pMB4698 arescreened for canthaxanthin production. Canthaxanthin-producingGRPBua[pMB4698] cells are transformed with pMB4692 and prototrophiccolonies are screened for astaxanthin production.

In another approach, the carotenoid ketolase and carotenoid hydroxylasegenes from P. marcusii can be introduced into the strains described inExample 2 above, in order to convert β-carotene into astaxanthin. P.marcusii genomic DNA is amplified with two primers.

CrtZfwd: 5′ CACACCGTCTCAAatgaccaatttcctgatcgtcgtc CrtZrev: 5′CACACAGATCtcacgtgcgctcctgcgcc,and the resulting fragment is cleaved with BsmBI, modified with theKlenow fragment of DNA polymerase, and cleaved with BglII. This fragmentis inserted into PmlI- and BamHI-cleaved pINA1269 (J. Mol. Microbiol.Biotechnol. 2 (2000): 207-216), containing the hp4d promoter, the XPR2terminator, the selectable LEU2 gene, and sequences necessary forselection and propagation in E. coli. The resulting plasmid “pA”contains sequences encoding carotene hydroxylase from P. marcusii (crtZgene) (Genbank accession: CAB56060.1) under the control of the hp4dpromoter.

“pYEG1TEF” is modified by substituting the LIP2 terminator for the XPR2terminator as follows. pINAl291 is digested with AvrII, modified withthe Klenow fragment of DNA polymerase, and cleaved with EcoRI, and thesmall LIP2t containing fragment is ligated to “pYEG1TEF” that has beendigested with SacII, modified with T4 DNA polymerase in the presence ofdNTP, and cleaved with EcoRI. The resulting plasmid is named“pYEGITEF-LIP2t”.

In order to amplify the carotenoid ketolase gene, P. marcusii genomicDNA is amplified with two primers.

CrtWfwd: 5′ CACACCCTAGGCCatgagcgcacatgccctgc CrtWrev: 5′CACACAAGCTTtcatgcggtgtcccccttg,and the resulting fragment is cleaved with AvrII and HindIII, andinserted into AvrII- and HindIII II-cleaved “pYEG1TEF-LIP2t”. Theresulting plasmid, “pBt”, contains sequences encoding the caroteneketolase (crtW gene) (Genbank accession: CAB56059.1) under the controlof the constitutive TEF1 promoter.

In order to combine the two expression cassettes into a single plasmid,“pBt” is cleaved with ClaI, modified with the Klenow fragment of DNApolymerase, and cleaved with EcoRI, and the crtW-containing fragment isisolated, mixed with the phosphorylated oligonucleotide adaptor pair:

5′ AATTCGCGGCCGCT and 5′ AGCGGCCGCG,cleaved with NotI, and ligated to NotI-digested “pA”. The resultingplasmid, “pABt”, contains both the TEF1p/crtW/LIP2t cassette and thehp4d/crtZ/XPR2t cassette as well as the selectable LEU2 gene.

“pABt” can be introduced into the Y. lipolytica strain described abovein Example 4 (TEF1p/a1-1/XPR2t; hp4d/carRP/LIP2t; GPDp/HMGR_(trunc)),and transformants selected for leucine prototrophy.

Example 7 Partial Inactivation of Y. Lipolytica Erg9 Gene EncodingSqualene Synthase Results in Increased Carotenoid Production

7A. In order to partially inactivate the ERG9 gene encoding squalenesynthase, the neighboring FOL3 gene is disrupted, resulting in a folinicacid requirement. This strain is then transformed with a mutagenizedfragment of DNA partially spanning the two genes, and For transformantsare screened for decreased squalene synthase activity.

The following oligonucleotides are synthesized:

PRIMER K 5′-CCTTCTAGTCGTACGTAGTCAGC; PRIMER L5′-CCACTGATCTAGAATCTCTTTCTGGand used to amplify a 2.3 kb fragment from Y. lipolytica genomic DNAspanning most of the FOL3 gene, using Pfu polymerase. The resultingfragment is cleaved with XbaI and phosphorylated, then ligated intopBluescriptSK⁻ that has been cleaved with KpnI, treated with T4 DNApolymerase (T4pol) in the presence of dNTPs, and subsequently cleavedwith XbaI. The resultant plasmid, designated pBS-fol3, is then cleavedwith Acc651 and EcoRI, treated with T4pol as above, and ligated to the3.4 kb EcoRV-SpeI ADE1 fragment (treated with T4pol) from pMB4529.

The resulting plasmid, pBSfol3Δade, can be cleaved with BsiWI and XbaIto liberate a 5.5 kb fragment that is used to transform the GRBPuastrains described above to adenine prototrophy. Resulting Ade⁺transformants are screened for a folinic acid requirement, and forhomologous integration by PCR analysis.

Strains that harbor the resultant fol3ΔADE1 allele can be transformedwith a 3.5 kb DNA fragment generated by mutagenic PCR amplificationusing the primers:

PRIMER M 5′-GGCTCATTGCGCATGCTAACATCG; PRIMER N5′-CGACGATGCTATGAGCTTCTAGACG,and Y. lipolytica genomic DNA as template. The resulting fragmentcontaining the N-terminal three-quarters of the FOL3 ORF and theC-terminal nine-tenths of the ERG9 ORF is used to transform strains. Theresulting Fol⁺Ade⁻ transformants are screened for decreased squalenesynthase activity by sensitivity to agents such as zaragozic acid,itraconazole, or fluconazole. Additionally, the resulting transformantsare screened for increased carotenoid production.

7B. Alternatively, the PCR fragment produced in 7A could be cloned andaltered in such a way as to remove the 3′-untranslated region of ERG9gene. Replacement of the fol3ΔADE1 disruption by this fragment resultsin decreased expression of squalene synthase [Schuldiner et al. (2005),Cell 123:507-519][Muhlrad and Parker (1999), RNA 5:1299-1307], which canbe confirmed as in 7A. This approach may also be used in a Fol⁺Ade⁻strain, using the ADE1 marker to disrupt the ERG9 3′-UTR.

7C. In still another approach, partially defective ERG9 alleles can beidentified in S. cerevisiae using plasmid shuffling techniques [Boeke etal. (1987), Methods Enzymol. 154:164-175], and using drug sensitivitiesas a phenotype. Defective genes can be transferred to Y. lipolyticausing standard molecular genetic techniques.

Example 8 Treatment of Y. Lipolytica Strains Producing Carotenoid withInhibitor of an Isoprenoid Biosynthesis Competitor Polypeptide Resultsin Increased Carotenoid Production

Cultures produced in Example 2 are treated with the squalene synthaseinhibitor, zaragozic acid (zaragozic acid at 0.5 μM) and monitored forβ-carotene production, as described above.

Example 9 Constructing an Oleaginous strain of Saccharomyces cerevisiae

The genes encoding the two subunits of ATP-citrate lyase from N. crassa,the AMP deaminase from Saccharomyces cerevisiae, and the cytosolic malicenzyme from M. circinelloides are overexpressed in S. cereviseae strainsin order to increase the total lipid content. Similar approaches toenhance lipid production could be employed in other host organisms suchas Xanthophyllomyces dendrorhous (Phaffia rhodozyma), using the same,homologous, or functionally similar oleaginic polypeptides.

Qiagen RNAEasy kits (Qiagen, Valencia, Calif.) are used to preparemessenger RNA from lyophilized biomass prepared from cultures of N.crassa. Subsequently, RT-PCR is performed in two reactions containingthe mRNA template and either of the following primer pairs.

acl1: 1fwd: 5′ CACACGGATCCTATAatgccttccgcaacgaccg 1rev: 5′CACACACTAGttaaatttggacctcaacacgaccc acl2: 2fwd: 5′CACACGGATCCAATATAAatgtctgcgaagagcatcctcg 2rev: 5′CACACGCATGCttaagcttggaactccaccgcac

The resulting fragment from the acl1 reaction is cleaved with SpeI andBamHI, and that from the acl2 reaction is cleaved with BamHI and SphI,and both are ligated together into YEp24 that has been digested withNheI and SphI, creating the plasmid “p12”. The bi-directional GAL1-10promoter is amplified from S. cerevisiae genomic DNA using the primers.

gal10: 5′ CACACGGATCCaattttcaaaaattcttactttttttttggatggac gal1: 5′CACACGGATCCttttttctccttgacgttaaagtatagagg,and the resulting 0.67 kb fragment is cleaved with BamHI and ligated ineither orientation to BamHI-digested “p12” to create “p1ga12” and“p2ga11”, containing GAL1-acl1/GAL10-acl2 and GAL10-acl1/GAL1-acl2,respectively (Genbank accession: acl1: CAB91740.2; acl2: CAB91741.2).

In order to amplify the S. cereviseae gene encoding AMP deaminase and apromoter suitable for expressing this gene, S. cerevisiae genomic DNA isamplified using two primer pairs in separate reactions:

AMD1 ORF: AMD1FWD: 5′ CACACGAGCTCAAAAatggacaatcaggctacacagag AMD1rev: 5′CACACCCTAGGtcacttttcttcaatggttctcttgaaattg GAL7p: gal7prox: 5′CACACGAGCTCggaatattcaactgtttttttttatcatgttgatg gal7dist: 5′CACACGGAtccttcttgaaaatatgcactctatatcttttag,and the resulting fragment from the AMD I reaction (2.4 kb) is cleavedwith SacI and AvrII, and that from the GAL7 reaction (0.7 kb) is cleavedwith BamHI and SphI, and both are ligated together into YEp13 that hasbeen digested with NheI and BamHI, creating the plasmid “pAMPD”. Thisplasmid carries the S. cerevisiae gene, AMDI, encoding AMP deaminase,under the control of the galactose-inducible GAL7 promoter.

Messenger RNA is prepared from lyophilized biomass of M. circinelloides,as described above, and the mRNA template is used in a RT-PCR reactionwith two primers:

MAEfwd: 5′ CACACGCTAGCTACAAAatgttgtcactcaaacgcatagcaac MAErev: 5′CACACGTCGACttaatgatctcggtatacgagaggaac,and the resulting fragment is cleaved with NheI and SalI, and ligated toXbaI- and XhoI-digested pRS413TEF (Mumberg, D. et al. (1995) Gene,156:119-122), creating the plasmid “pTEFMAE”, which contains sequencesencoding the cytosolic NADP⁺-dependant malic enzyme from M.circinelloides (E.C. 1.1.1.40; mce gene; Genbank accession: AY209191)under the control of the constitutive TEF1 promoter.

The plasmids “p1ga12”, “pAMPD”, and “pTEFMAE” are sequentiallytransformed into a strain of S. cereviseae to restore prototrophy foruracil (“p1ga12”), leucine (“pAMPD”), and histidine (“pTEFMAE”) (Guthrieand Fink Methods in Enzymology 194:1-933, 1991). The resultingtransformants are tested for total lipid content following shake flasktesting in either synthetic complete (SC) medium lacking uracil, leucineand histidine, as described in Example 3, or in a 2-step fermentationprocess. In the 2-step process, 1.5 ml of cells from an overnight 2 mlroll tube culture containing SC medium lacking uracil, leucine andhistidine are centrifuged, washed in distilled water, and resuspended in20 ml of a nitrogen-limiting medium suitable for lipid accumulation (30g/L glucose, 1.5 g/L yeast extract, 0.5 g/L NH₄Cl, 7 g/L KH₂PO₄, 5 g/LNa₂HPO₄-12H₂O, 1.5 g/L MgSO₄-7H₂O, 0.08 g/L FeCl₃-6H₂O, 0.01 g/LZnSO₄-7H₂O, 0.1 g/L CaCl₂-2H₂O, 0.1 mg/L MnSO₄-5H₂O, 0.1 mg/LCuSO₄-5H₂O, 0.1 mg/L Co(NO₃)₂-6H₂O; pH 5.5 (J Am Oil Chem Soc 70:891-894(1993)).

Intracellular lipid content of the modified and control S. cerevisiaestrains is analyzed using the fluorescent probe, Nile Red (J MicrobiolMeth (2004) 56:331-338). In brief, cells diluted in buffer are stainedwith Nile Red, excited at 488 nm, and the fluorescent emission spectrain the wavelength region of 400-700 nm are acquired and compared to thecorresponding spectra from cells not stained with Nile Red. To confirmresults from the rapid estimation method, the total lipid content isdetermined by gas chromatographic analysis of the total fatty acidsdirectly transmethylesterified from dried cells, as described (ApplMicrobiol Biotechnol. 2002 November; 60(3):275-80). Non-transformed S.cerevisiae strains produce 6% and 10% total lipid (dry cell weightbasis) after growth in YPD and lipid accumulation medium, respectively.Yeast strains expressing the multiple oleaginic polypeptides produce 17%and 25% total lipid following growth in YPD and lipid accumulationmedium, respectively.

Example 10 Introduction of Heterologous Carotene Hydroxylase into Y.Lipolytica Strains Producing Carotenoid for Production of Zeaxanthin

MF578 (tef-carRP tef-carB) was transformed with pMB4692 that had beencleaved with SalI. Several Ura⁺ colonies inferred to contain tef-crtZ byPCR analysis were able to produce zeaxanthin in YPD shake flasks, and inone case, all of the carotene was depleted.

The following tables are referenced throughout the description:

TABLE 1 Examples of acetyl-CoA carboxylase polypeptides. Genbank RowACCESSION Genbank GI 1 XP_410263 49097606 2 XP_329580 32418204 3XP_386756 46124405 4 XP_367702 39972623 5 XP_501721 50548503 6 EAK9970846440402 7 XP_457211 50413128 8 NP_982612 45184894 9 XP_449236 5029364910 NP_593271 19114183 11 NP_014413 6324343 12 XP_455355 50310667 13T42531 11272737 14 AAA20073 171504 15 EAL20176 50257469 16 XP_57131658268320 17 XP_402244 49076566 18 S60200 2133343 19 BAA24410 2804173 20P32874 1708192 21 S55089 7438088 22 NP_990836 45382859 23 CAE0147132526576 24 AAR37018 40019048 25 NP_001... 57164283 26 NP_77664927806341 27 CAI25271 56205878 28 XP_109883 51828611 29 NP_94213438679971 30 NP_942131 38679960 31 NP_942135 38679974 32 NP_94213638679977 33 AAP94122 33112885 34 NP_071529 11559962 35 2006242A 74096436 AAS13685 42405896 37 NP_598665 48976025 38 Q13085 2493311 39XP_548250 57091783 40 XP_314071 58385597 41 CAG08536 47226520 42NP_724636 24586460 43 NP_610342 24586458 44 NP_001084 4501855 45NP_446374 16758804 46 EAL63219 60465120 47 NP_921034 37533464 48 T070847438099 49 AAP78896 32264940 50 AAO62903 29123370 51 BAA07012 1100253 52AAL02056 15558947 53 AAG40563 11869927 54 D86483 25293894 55 T079207438090 56 A57710 2130099 57 AAO62902 29123376 58 2208491A 1588584 59T09538 7438102 60 CAC19875 12057067 61 AAP78897 32264942 62 T022357438095 63 AAG40564 11869928 64 E86483 25293893 65 CAC84161 20975574 66T07081 7438097 67 CAC19876 12057069

TABLE 2 Examples of pyruvate decarboxylase polypeptides. Genbank RowACCESSION Genbank GI 1 1QPBB 7245977 2 CAA54522 871533 3 1PYDB 515237 4CAA28380 4109 5 1PVDB 1127233 6 CAA33709 4114 7 AAN77243 25992752 8NP_013235 6323163 9 Q6FJA3 57012668 10 S36363 486942 11 Q12629 5278827912 AAP75898 37359468 13 S70684 2131152 14 NP_011601 6321524 15 AAQ7361834500072 16 NP_983270 45185554 17 AAF78895 8745337 18 CAB65554 668966219 AAP75899 37359470 20 NP_982469 45184751 21 CAA97091 1945321 22 S507001086157 23 XP_446491 50288125 24 XP_462338 50427451 25 AAC03164 1706678426 EAK96569 46437219 27 XP_457131 50412425 28 AAC03165 2734883 29XP_459224 50421349 30 CAH56494 52673248 31 XP_502647 50550349 32NP_010203 6320123 33 BAA04886 1786148 34 XP_449074 50293325 35 EAL0409846444826 36 CAD60727 27803024 37 T38759 25777585 38 XP_331173 3242145939 NP_594083 19114995 40 XP_401609 49075036 41 XP_390010 46136637 42XP_409025 49095128 43 NP_984350 45188127 44 AAD16178 4323053 45 P872082501326 46 EAL18331 50255598 47 XP_567475 58260130 48 AAM73540 2166601149 AAM73539 21666009 50 XP_502508 50550071 51 CAA93158 1177659 52XP_412533 49123327 53 P51844 1706333 54 XP_455842 50311631 55 CAA611553688422 56 XP_444902 50284947 57 CAA47319 4118

TABLE 3 Examples of isocitrate dehydrogenase polypeptides. Genbank RowACCESSION Genbank GI 1 O13285 3023996 2 EAK91676 46432179 3 O132853023996 4 EAK94305 46434909 5 XP_451683 50303483 6 XP_459772 50422415 7O13294 27805482 8 XP_460289 50423413 9 XP_390523 46137663 10 XP_36734339971905 11 XP_323176 32405126 12 XP_445447 50286037 13 AAK7673015027826 14 NP_010217 6320137 15 NP_984921 45190667 16 AAK76731 1502782717 P79089 3023999 18 NP_013275 6323203 19 XP_407136 49091350 20NP_982520 45184802 21 XP_446953 50289047 22 XP_445184 50285511 23XP_455638 50311227 24 AAA64516 736722 25 NP_970434 42525054 26 AAT9317351013759 27 XP_569233 58264154 28 XP_569234 58264156 29 XP_40372649080406 30 XP_503571 50552322 31 XP_458151 50428131 32 O13302 1312430133 XP_409927 49096934 34 XP_385909 46122711 35 XP_365293 39967489 36NP_983873 45187650 37 XP_455266 50310493 38 NP_594397 19115309 39XP_324955 32408949 40 CAE81942 38636405 41 NP_014361 6324291 42XP_446479 50288101 43 XP_567378 58259936 44 XP_398944 49069310 45XP_502479 50550013 46 EAK96238 46436883 47 EAK96305 46436951 48XP_461797 50426401 49 XP_328403 32415850 50 CAF31997 42820684 51XP_389756 46136129 52 XP_363786 39952139 53 AAL73035 18463935 54XP_405140 49086142 55 NP_595203 19111995 56 NP_014779 6324709 57XP_447564 50290265 58 NP_985684 45198655 59 XP_566837 58258849 60XP_454086 50308171 61 XP_398943 49069308

TABLE 4 Examples of ATP-citrate lyase polypeptides. Genbank RowACCESSION Genbank GI 1 XP_327071 32413182 2 O93988 30912679 3 XP_37022239977669 4 XP_406573 49090008 5 XP_504787 50554757 6 Q9P7W3 30912748 7XP_398620 49068662 8 NP_596202 19112994 9 XP_567460 58260100 10NP_001008 56118260 11 XP_418154 50760837 12 AAH84253 54038148 13NP_942127 38569423 14 NP_001087 38569421 15 P53396 20141248 16 AAL3431617028103 17 NP_001002 50540366 18 AAH84776 54311201 19 S21173 105392 20AAT94429 51092031 21 AAD34754 28372804 22 AAH21502 18204829 23 XP_31932358392375 24 NP_725514 24653990 25 EAL26601 54637198 26 CAE56725 3957941927 CAE64663 39593194 28 XP_511495 55645405 29 CAF95829 47210997 30AAO22565 27754223 31 AAL33788 17065616 32 CAB46077 5304837 33 CAF9604447204726 34 AAK13318 13160653 35 AAQ75159 34558815 36 AAQ75128 3455878337 XP_537640 57091075 38 XP_327069 32413178 39 CAB76164 7160184 40XP_370223 39977671 41 XP_386215 46123323 42 CAA10666 7159697 43XP_406572 49090004 44 XP_503231 50551515 45 NP_593246 19114158 46XP_398620 49068662 47 XP_567460 58260100 48 AAT94429 51092031 49NP_725514 24653990 50 AAD34754 28372804 51 EAL26601 54637198 52XP_319323 58392375 53 AAH84776 54311201 54 BAB00624 9229902 55 NP_00100856118260 56 AAH84253 54038148 57 AAH56378 38614162 58 NP_001087 3856942159 NP_942127 38569423 60 P53396 20141248 61 XP_511495 55645405 62NP_058683 8392839 63 NP_001002 50540366 64 S21173 105392 65 NP_50828017551266 66 CAE64663 39593194 67 CAE56725 39579419 68 NP_506267 1755734469 XP_537640 57091075 70 CAF96059 47204551 71 F96633 25404292 72AAM91141 22136126 73 NP_849634 30681854 74 AAO23582 27764922 75 AAM6507821593129 76 CAC86996 15919089 77 AAQ75158 34558814 78 AAQ75127 34558782

TABLE 5 Examples of malic enzyme polypeptides. Genbank Row ACCESSIONGenbank GI 1 NP_012896 6322823 2 XP_448858 50292851 3 XP_454793 503095634 NP_986598 45201028 5 XP_460887 50424595 6 EAK97738 46438407 7XP_504112 50553402 8 XP_330094 32419237 9 XP_380981 46107844 10XP_411070 49102552 11 XP_362875 39946676 12 NP_587760 19075260 13NP_978189 42780942 14 YP_035982 49481098 15 YP_027934 49184682 16YP_018438 47527089 17 ZP_002365 47565532 18 YP_083209 52143619 19XP_571672 58269032 20 NP_391586 16080758 21 YP_092693 52786864 22NP_831516 30019885 23 YP_093460 52787631 24 YP_081030 52082239 25NP_822689 29828055 26 O34389 33517449 27 EAL19111 50256386 28 NP_82504729830413 29 ZP_002340 47096498 30 NP_928837 37525493 31 NP_23083315641201 32 NP_934257 37679648 33 NP_761613 27366085 34 AC1314 2528368835 YP_055602 50842375 36 YP_095310 52841511 37 ZP_002315 47093832 38AC1686 25283689 39 YP_126594 54294179 40 YP_123567 54297198 41 EAJ7626044510091 42 YP_114273 53803890 43 NP_797637 28898032 44 YP_04025049483026 45 ZP_001276 53693400 46 YP_044961 50083451 47 YP_12822654295811 48 NP_719387 24375344 49 XP_572853 58271394 50 NP_25216115598667 51 ZP_001368 46164263 52 YP_125345 54298976 53 NP_79369528871076 54 YP_096964 52843165 55 EAH92280 44245125 56 YP_15498856459707 57 EAI68195 44354928 58 YP_070054 51595863 59 YP_13302554303032 60 NP_969623 42524243 61 NP_856009 31793516 62 DECARBOXYATING)) 63 NP_935035 37680426 64 YP_050922 50121755 65 E70705 7431223 66NP_216848 57116971 67 DECARBOXY ATING)) 68 YP_143786 55980489 69YP_130202 54309182 70 NP_415996 16129438 71 NP_819843 29654151 72NP_753809 26247769 73 NP_707611 56479957 74 F85728 25283682 75 YP_16369056552851 76 YP_150562 56413487 77 NP_720610 24378655 78 NP_46052516764910 79 ZP_003193 48865537 80 NP_784797 28377905 81 T13496 743122782 AAV65766 55793550 83 A97096 25283683 84 YP_193951 58337366 85 H9709625283684 86 ZP_003237 48870993 87 ZP_001460 41689468 88 D86737 2528367689 ZP_002870 48825851 90 ZP_001439 34762975 91 1922245A 737262 92YP_169914 56708018 93 YP_055027 50841800 94 ZP_000625 23023297 95NP_296302 15807565 96 NP_285599 15807938 97 YP_132069 54302076 98CAA50716 467569 99 ZP_002906 48833596 100 ZP_003155 48861632 101NP_773109 27381580 102 AAQ95658 37622953 103 CAC19505 56204311 104AAH80660 51873855 105 P40927 729986 106 AAT02533 46850200 107 BAC3708626346875 108 T02763 7431235 109 XP_387367 46125627 110 AAC50613 1465733111 CAA39421 669118 112 CAA39420 669117 113 NP_032641 6678912 114CAA39419 581228 115 AAB01380 1335389 116 JC4160 1085347 117 E9682825283677 118 BAD87910 57899974 119 EAJ77083 44511304 120 P13697 266504121 NP_036732 7106353 122 YP_065939 51246055 123 CAC18164 16944467 124XP_322953 32404680 125 AAK91502 18460985 126 AAQ88396 37147841 127NP_001003 57525624 128 1GQ2P 21465488 129 AAO26053 28195290 130 AAH8425054038006 131 XP_362590 39946106 132 AAH03287 13096987 133 Q29558 2497785134 XP_532217 57094622 135 P28227 126734 136 NP_496968 17537199 137NP_914533 34906372 138 AAD10504 4096786 139 AAO67523 50897495 140 P432791170871 141 AAK83074 15077109 142 AAP33011 30575690 143 AAN8669027357017 144 P78715 41017288 145 AAP32204 30526303 146 AAV31249 54287505147 T06402 7431232 148 Q99KE1 55583978 149 XP_399922 49071266 150 P36444547886 151 AAO30034 28059162 152 AAK83073 15077107 153 NP_002387 4505145154 AAA33487 168528 155 BAA74735 4239891 156 NP_989634 45383538 1571GZ3D 31615316 158 AAW56450 57791240 159 AAT02534 46850202 160 S29742422339 161 1O0SB 34811253 162 P27443 126732 163 T06401 7431231 164AAL16175 16226466 165 AAF73006 8118507 166 AAK97530 15420975 167EAI90348 44385841 168 P51615 1708924 169 AAA19575 169327 170 S437181084300 171 P34105 1346485 172 AAS38597 42733630 173 BAC54101 27530932174 AAT02535 46850204 175 CAB66003 6706333 176 AAH84860 54311418 177CAA39422 669119 178 NP_916713 34910732 179 CAA56354 510876 180 DEFBC7427668 181 JC5967 7431234 182 NP_197960 15239517 183 NP_651959 21356279184 CAB64263 6634090 185 BAB20887 54606800 186 EAL27424 54638022 187NP_006671 5729920 188 AAB08874 1561774 189 1PJLH 33358128 190 1GZ4D22218682 191 1QR6B 5822327 192 1PJ3D 39654475 193 P22178 126736 194XP_410305 49097690 195 AAH22472 18490280

TABLE 6 Examples of AMP deaminase polypeptides. Genbank Row ACCESSIONGenbank GI 1 AAA34420 171053 2 XP_446684 50288509 3 NP_983153 45185436 4XP_453337 50306727 5 EAL02322 46443037 6 XP_460211 50423261 7 XP_50382250552824 8 XP_413009 49131023 9 XP_360256 39941438 10 XP_381547 4610897811 XP_330167 32419447 12 CAB97316 16945394 13 T50996 11359582 14NP_595153 19111945 15 EAL22226 50259553 16 XP_402237 49076548 17CAA62797 995562 18 AAF65407 7638159 19 XP_537039 57088163 20 AAH4911929145073 21 XP_569691 58265070 22 AAD56303 5922018 23 NP_004028 2126431824 A44313 345738 25 CAI19307 56206061 26 AAA62126 644509 27 CAI1930556206059 28 XP_310497 58424203 29 CAI19306 56206060 30 AAC50308 60849931 CAG06825 47229629 32 NP_727741 45551453 33 NP_727739 45551452 34NP_727740 24641890 35 AAN09337 22832227 36 T01259 7484807 37 XP_50659151963676 38 NP_850294 30687456 39 CAG07509 47228777 40 NP_49497432564190 41 T15771 7497030 42 CAE59064 39596837 43 NP_494973 32564194 44BAA06505 1321635 45 NP_000471 4502079 46 S68147 2134756 47 AAH5638038614134 48 O08739 2494043 49 NP_113732 13928736 50 O09178 2494044 51XP_420973 50747746 52 NP_956142 41054127 53 CAG01709 47222742 54NP_957187 41053780 55 XP_392957 48104570 56 AAH07183 13938134 57CAG05605 47220579 58 NP_620231 20302047 59 XP_540247 57098851 60CAF99638 47230445 61 XP_513671 55587796 62 CAI18828 56203368 63 CAI1882956203369 64 CAI18830 56203370 65 EAA19931 23484684 66 CAH99706 5650093267 XP_131103 38076931 68 CAH77387 56523366

TABLE 7 Examples of acetoacetyl-CoA thiolase polypeptides. Genbank RowACCESSION Genbank GI 1 P10551 135758 2 Q04677 418002 3 Q12598 34925109 4T10247 7433657 5 T42741 11257345 6 AAL18924 16417944 7 AAM67058 216180088 AAO51605 28829030 9 AAU95618 53854350 10 AAU95619 53854352 11 BAA970038777413 12 CAE76429 38567134 13 EAK90852 46431255 14 EAL32264 5464352015 NP_015297 6325229 16 NP_568694 30695411 17 NP_572414 24640423 18NP_596686 19113478 19 NP_851154 30695409 20 NP_908411 34894172 21NP_974900 42573608 22 NP_974901 42573610 23 NP_984262 45188039 24XP_389497 46134945 25 XP_401186 49074048 26 XP_405546 49087148 27XP_449306 50293789 28 XP_449306 50293789 29 XP_450298 50899020 30XP_453599 50307241 31 XP_460741 50424309 32 XP_500646 50546253

TABLE 8 Examples of HMG-CoA synthase polypeptides. Genbank Row ACCESSIONGenbank GI 1 B55729 1083370 2 P54869 1708235 3 S13887 86312 4 S27197284048 5 AAA37076 387072 6 AAF89580 9621905 7 AAH00297 33991031 8AAH31363 21618633 9 AAH42929 27552834 10 AAH79694 50925193 11 AAH8354354035469 12 AAO52569 28830079 13 AAP35966 30583443 14 BAB23657 1283643915 BAC04559 21754758 16 BAC05233 21758044 17 CAA52032 1772495 18CAC18553 11602786 19 CAG33131 48145817 20 CAH92111 55730782 21 CAI2240856205097 22 EAK97451 46438115 23 EAL25034 54635631 24 NP_002121 5402072025 NP_013580 6323509 26 NP_032282 31560689 27 NP_058964 8393538 28NP_593859 19114771 29 NP_666054 31981842 30 NP_725570 24654139 31NP_775117 27465521 32 NP_957379 41055180 33 NP_983739 45187516 34NP_990742 45382279 35 NP_999545 47523816 36 XP_315872 58387870 37XP_323241 32405256 38 XP_368218 39973655 39 XP_389442 46134253 40XP_397202 48141273 41 XP_402977 49078452 42 XP_409060 49095198 43XP_446972 50289085 44 XP_453529 50307101 45 XP_456470 50405663 46XP_506052 50557288 47 XP_513693 55587844 48 XP_536483 57085299 49XP_569805 58265298 50 XP_571930 58269548

TABLE 9 Examples of HMG-CoA reductase polypeptides. Genbank RowACCESSION Genbank GI 1 A23586 90238 2 O74164 11132850 3 P51639 1708252 4P54960 1708251 5 Q12649 18276268 6 Q29512 2495262 7 Q9Y7D2 11133211 8S30338 422383 9 S72194 7450066 10 AAA36989 387052 11 AAA37077 305355 12AAA49740 214237 13 AAD20975 9817458 14 AAH74197 49257596 15 AAL0935115824453 16 AAO85434 29468180 17 AAP72015 32165622 18 AAR02862 4527211819 AAT92819 51013051 20 BAC20567 23574646 21 CAA63970 4376229 22CAE47850 41581201 23 CAF92135 47213283 24 CAH92577 55731745 25 EAK9457746435190 26 EAL20195 50257490 27 AAF80374 8886086 28 NP_013555 632348329 NP_013636 6323565 30 NP_032281 56119096 31 NP_037266 40538852 32NP_588235 19075735 33 NP_985010 45190756 34 NP_989816 45383193 35NP_999724 47551099 36 XP_324892 32408825 37 XP_364130 39955070 38XP_389373 46134115 39 XP_400629 49072680 40 XP_405730 49087632 41XP_407954 49092986 42 XP_449268 50293713 43 XP_451740 50303597 44XP_458872 50420671 45 XP_503558 50552167 46 XP_536323 57084803 47XP_571450 58268588

TABLE 10 Examples of mevalonate kinase polypeptides. Genbank RowACCESSION Genbank GI 1 XP_386088 46123069 2 XP_408006 49093090 3XP_370449 39978123 4 EAL04797 46445529 5 XP_322935 32404644 6 NP_00100755925207 7 XP_460851 50424525 8 XP_567851 58260882 9 XP_567850 5826088010 AAQ02416 33303805 11 CAA53059 450346 12 AAH16140 16359371 13 AAH0560613542811 14 XP_403111 49078786 15 XP_452532 50305147 16 CAG0852747226511 17 XP_446138 50287417 18 AAO51522 28828936 19 NP_98519145190937 20 XP_500956 50546973 21 NP_013935 6323864 22 AAD45421 557871823 NP_920723 37532842 24 NP_851084 30690651 25 AAL18925 16417946 26NP_788338 28573850 27 AAU20834 51988124 28 AAU87813 52839819 29 AAU2083551988125 30 YP_183887 57641409 31 NP_143478 14591399 32 BAA24409 280417233 NP_126232 14520757 34 XP_522574 55639331 35 NP_071114 11499870 36XP_423949 50797461 37 NP_633786 21227864 38 ZP_002971 48840229 39EAH50787 44170778 40 NP_615566 20089491 41 1VISA 40890012 42 EAK0355944549994 43 NP_248080 15669275 44 1KKHA 20150886 45 Q50559 2497518 46CAF88123 47200914 47 NP_275189 15678075 48 EAI88745 44383877 49ZP_002040 46141948 50 XP_543435 57105916 51 EAI38920 44313360 52NP_148611 14602065 53 EAD08953 43286228 54 EAD45697 43361720 55YP_134862 55377012 56 NP_720650 24378695 57 NP_614276 20094429 58 E8427025409931 59 NP_691146 23097680 60 ZP_003233 48870579 61 AAG02440 993738662 EAD12278 43292898 63 NP_498328 17555862 64 EAB31483 42928976 65ZP_003319 50590618 66 NP_814642 29375488 67 AC1434 25514495 68 ZP_00357753796847 69 EAD82048 43454743 70 CAE73618 39586491 71 YP_012624 4690623572 NP_988455 45358898 73 ZP_002348 47097293 74 ZP_002862 48824993 75ZP_002307 47093020 76 NP_597102 19173299 77 CAD24422 20429111 78NP_785308 28378416 79 EAA39098 29247539 80 NP_819638 29653946 81EAH49746 44168765 82 EAH49745 44168764 83 NP_378182 15922513 84ZP_000459 23002259 85 H90181 25393827 86 YP_054120 50405028 87 BAB077909695270 88 AAG02435 9937379 89 NP_560495 18313828 90 YP_187834 5786618791 EAK40782 44602942 92 CAC51370 15212070 93 AAG02424 9937364 94YP_185521 57651465 95 YP_040044 49482820 96 YP_194037 58337452 97 D8667525400965 98 NP_763916 27467279 99 CAF89434 47197810 100 EAF3833343767792 101 EAK46841 44611394 102 H89827 25507776 103 ZP_00314948861061 104 EAK17824 44570143 105 EAH86276 44235719 106 YP_11841854024176 107 ZP_003196 48865749 108 AAG02430 9937372 109 NP_26907515674901 110 NP_802520 28896170 111 AAL97579 19748102 112 ZP_00366656808907 113 NP_965060 42519130 114 NP_819639 29653947 115 EAD9702443484567 116 BAD86800 57753870

TABLE 11 Examples of phosphomevalonate kinase polypeptides. Genbank RowACCESSION Genbank GI 1 AAA34596 171479 2 XP_452514 50305111 3 NP_98521045190956 4 XP_446144 50287429 5 XP_462340 50427455 6 EAL04096 46444824 7EAL03941 46444668 8 XP_503619 50552418 9 XP_389940 46136497 10 XP_32979532418634 11 XP_369652 39976529 12 XP_406448 49089559 13 NP_59342119114333 14 XP_568385 58261950 15 EAL17628 50254887 16 AAL18926 1641794817 BAD43274 51969164 18 BAD44652 51971975 19 XP_398375 49068172 20BAD44486 51971643 21 F90479 25393214 22 YP_194039 58337454

TABLE 12 Examples of mevalonate pyrophosphate decarboxylasepolypeptides. Genbank Row ACCESSION Genbank GI 1 AAT93171 51013755 21FI4A 13786942 3 XP_455548 50311049 4 XP_445335 50285813 5 XP_45691250409853 6 NP_986435 45200865 7 AAF19399 6625790 8 XP_328845 32416734 9XP_505041 50555265 10 NP_594027 19114939 11 XP_364905 39963452 12XP_390600 46137817 13 XP_408551 49094180 14 AAA34506 7544604 15 EAL1892750256200 16 XP_568247 58261674 17 XP_402794 49077992 18 AAH8178451980639 19 EAL00166 46440864 20 NP_619597 20149736 21 NP_11232413592005 22 BAC40852 26354448 23 XP_546783 57087071 24 Q99JF5 2381409525 AAH63907 39645379 26 CAF99534 47230341 27 AAP35576 30582699 28AAP36301 30584105 29 AAL18927 16417950 30 AAV32433 54292590 31 AAP6820831711704 32 AAM64988 21593039 33 NP_566995 18410026 34 XP_42313050771155 35 AAM65192 21593243 36 NP_001007 55925435 37 NP_57306828571205 38 BAD27942 50252009 39 T47584 11281655 40 XP_307373 3119685141 CAE73245 39591192 42 NP_496966 17537201 43 XP_393230 48121058 44G90479 25393662 45 NP_496967 17537203 46 NP_691147 23097681 47 EAL2928254640164 48 AD1434 25515042 49 ZP_002308 47093021 50 YP_012625 4690623651 ZP_002348 47097294 52 NP_819637 29653945 53 NP_376888 15921219 54ZP_003319 50590617 55 NP_585805 19074299 56 YP_187835 57866188 57CAD24423 20429112 58 AAG02431 9937373 59 NP_763917 27467280 60 AAG024469937394 61 ZP_002863 48824994 62 AAG02441 9937387 63 YP_185522 5765146664 A89828 25505863 65 NP_814641 29375487 66 YP_040045 49482821 67NP_785307 28378415 68 ZP_003196 48865750 69 ZP_003233 48870580 70 E8667525400967 71 EAE31110 43552684 72 BAB07791 9695271 73 CAC51371 1521207174 ZP_000459 23002258 75 NP_965061 42519131 76 BAD86801 57753871 77YP_194038 58337453 78 YP_118419 54024177 79 EAK18820 44571499 80EAI85935 44379784 81 NP_721336 24379381 82 D95044 25388338 83 AAG024569937408 84 C97914 25511486 85 EAK47683 44612560 86 EAB86425 43039778 87YP_140971 55822530 88 YP_139081 55820639 89 BAD07376 40882372 90NP_968512 42523132 91 EAI06705 44265427 92 YP_060018 50914046 93AAG02451 9937401 94 NP_269076 15674902 95 ZP_003666 56808906 96NP_688323 22537472 97 NP_735832 25011437 98 EAC40267 43149093 99AAL97580 19748103 100 EAI76915 44367119 101 EAD35042 43339207 102YP_073129 51598941 103 EAI90092 44385501 104 BAB07818 9711347 105EAD72850 43433025 106 NP_212820 15595031 107 YP_124337 54297968 108YP_096056 52842257 109 EAA39903 29248368 110 EAH06252 44088237 111YP_127354 54294939 112 EAD45753 43361830 113 NP_802519 28896169

TABLE 13 Examples of IPP isomerase polypeptides. Genbank Row ACCESSIONGenbank GI 1 NP_015208 6325140 2 XP_448008 50291151 3 NP_983828 451876054 XP_455121 50310203 5 XP_462358 50427491 6 EAL01685 46442395 7XP_504974 50555131 8 XP_328425 32415894 9 XP_367200 39971619 10XP_389898 46136413 11 XP_404716 49085144 12 CAD37150 21627818 13NP_595164 19111956 14 XP_566641 58258457 15 XP_402453 49077100 16 O355866225528 17 AAP36609 30584713 18 AAF37873 7188790 19 NP_445991 1675830620 O42641 6225529 21 BAA33979 3790386 22 Q13907 6225527 23 AAH2241848257241 24 AAH19227 48257312 25 AAH57827 35505325 26 NP_004499 4001863327 AAH89786 58477715 28 CAH91844 55730243 29 XP_418561 50732281 30AAH06999 48257093 31 CAF98782 47225155 32 NP_808875 29366820 33XP_507622 55633353 34 AAH82648 52139082 35 NP_001011 58332496 36AAF29976 6856556 37 AAG10423 9971806 38 O48964 6225525 39 AAF299736856550 40 AAF29977 6856558 41 AAQ84167 35186998 42 AAF29974 6856552 43Q39472 6225526 44 S49588 1085973 45 AAL91980 19568939 46 BAB4097313603406 47 AAF29975 6856554 48 T52027 25493162 49 AAL91979 19568937 50T46812 11362218 51 T51248 11362217 52 BAB40974 13603408 53 O489656225532 54 XP_225509 34877710 55 XP_506401 51963472 56 AAF29978 685656057 AAH76541 50369278 58 AAT94033 51038230 59 XP_225502 34876517 60Q39471 6225533 61 AAB67743 1213450 62 NP_197148 22326844 63 BAB096119759126 64 AAD41766 5305669 65 AAB67741 1213442 66 XP_395125 48101420 67AAN28784 23505849 68 AAF36996 7110585 69 BAB16690 15289752 70 AAQ1486933340598 71 BAC65421 28971819 72 S71369 2129625 73 AAF29979 6856562 74AAF29980 6856564 75 AAP21674 30267831 76 Q39664 6225534 77 NP_65096224648688 78 AAM50284 21429130 79 XP_321388 58395620 80 Q9BXS1 2097850681 T07979 7484383 82 XP_225508 34876527 83 AAT92102 51011386 84XP_225507 34876555 85 XP_344623 34876537 86 S44843 630677 87 XP_22549827687955 88 AAT08468 47013849 89 EAI79636 44370808 90 CAE75055 3958740191 EAL04047 46444775 92 XP_225528 34876543 93 XP_544282 57040602 94XP_225511 27688013 95 P26173 114853 96 EAJ04069 44405322 97 EAH2749644127513 98 AAF91499 9653280 99 AAM48661 21328655 100 EAK17826 44570145101 EAD59515 43391069 102 YP_128702 54307682 103 EAK66656 44639203 104YP_118189 54023947 105 T50740 11282665 106 ZP_002077 46193541 107EAK16470 44568229 108 YP_165403 56695056 109 EAD08775 43285885 110YP_195623 58616494 111 EAI38918 44313358 112 NP_930583 37527239 113YP_160254 56478665 114 EAH69842 44206571 115 EAK26254 44582307 116AAR24381 38569721 117 AAM48607 21328600 118 EAD82049 43454744 119ZP_001924 45914126 120 YP_056780 50843553 121 YP_050880 50121713 122EAF29235 43749645 123 NP_630823 21225044 124 Q82MJ7 34582349 125ZP_003374 52010110 126 AAS75819 45737905 127 Q8KP37 30913023 128XP_507621 55633351 129 XP_344621 34876521 130 XP_346322 34880719 131YP_152060 56414985 132 AAT42442 48429280 133 Q9KK75 13878536 134NP_806649 29143307 135 YP_063124 50955836 136 Q8FND7 46395593 137CAF20647 41326485 138 Q8NN99 23821718 139 Q7X5H2 46395586 140 NP_33624615841209 141 Q83MJ9 46395588 142 P60923 46395576 143 Q8FE75 31563050 1441R67A 38493022 145 Q9KWD1 13878537 146 Q7VEU0 46395585 147 B8433325410326 148 NP_417365 16130791 149 E85944 25355426 150 1HZTA 15826050151 1PVFB 50513321 152 EAD63579 43403471 153 1I9AB 13786886 154YP_012992 46906603 155 ZP_002293 47091503 156 EAI37194 44310821 157YP_137864 55380014 158 CAD92056 42516867 159 1OW2B 42543244

TABLE 14 Examples of FPP synthase polypeptides. Genbank Row ACCESSIONGenbank GI 1 Q92250 2497455 2 XP_363065 39948036 3 XP_386960 46124813 4Q92235 3122099 5 XP_412149 49116518 6 XP_503599 50552378 7 NP_59329919114211 8 CAD42869 21955860 9 XP_448787 50292709 10 NP_012368 632229411 T42081 7433997 12 EAK93751 46434339 13 XP_451300 50302727 14XP_571137 58267962 15 XP_460720 50424267 16 NP_984739 45190485 17BAD15361 46367743 18 S71433 7433991 19 CAA65643 1523990 20 XP_39906149069544 21 S71432 7433990 22 AAH68912 46249832 23 1FPS 1065289 24P08836 3915686 25 AAH83515 53733369 26 1UBX 1942050 27 1UBY 1942051 28AAF37872 7188788 29 NP_803463 29135293 30 AAK63847 14488053 31 AAV5889655710092 32 T06272 7433988 33 JC4846 2117737 34 P05369 120478 35 O2424125452945 36 O24242 25452946 37 AAH59125 37590777 38 AAH48497 28913418 39AAP74720 32329199 40 CAG11850 47225367 41 AAM51429 21436457 42 AAP7471932329197 43 AAM08927 20135548 44 XP_537252 57089113 45 AAQ56011 3401369246 AAQ14872 33340604 47 AAQ14871 33340602 48 AAD17204 4324960 49AAH87886 56789674 50 AAK68152 14573639 51 AAA52423 182399 52 S664702129849 53 CAA29064 4725 54 CAI12715 55957735 55 BAA03523 40788949 56P14324 1346031 57 S66471 2129850 58 AAA35820 182405 59 CAA59170 149164160 BAB16687 15289750 61 CAA72793 1922251 62 CAH91070 55728661 63AAK58594 14279425 64 AAB07264 1146159 65 Q09152 21431776 66 O649056016044 67 BAB60822 14422406 68 S52009 1076319 69 NP_917118 34911542 70AAD32648 4894899 71 AAA40960 203582 72 AAR27053 38684029 73 AAU4399852353430 74 AAL82595 18958450 75 NP_917069 34911444 76 XP_22880234879769 77 BAD81810 56785155 78 AAN62522 24796660 79 NP_595334 1911212680 T52066 25458583 81 AAL49067 17946048 82 CAA08919 3395483 83 XP_54766257089869 84 EAL26135 54636732 85 BAB60821 14422404 86 AAP74721 3232920187 XP_496902 51466663 88 XP_474182 50929309 89 CAA87327 1160178 90BAD20729 47776234 91 BAC53873 30984142 92 BAB69490 15991313 93 NP_97456542572937 94 CAA08918 5678609 95 AAP86267 32527731 96 AAO17735 3052295397 AAK71861 14647139 98 AAL73357 18478919 99 AAO63552 29124957 100CAI00471 56498227 101 NP_701155 23508486 102 XP_474180 50929305 103AAL73358 18478922 104 EAH48995 44167328 105 NP_493027 17508563 106CAE71711 39580204 107 XP_487220 51766977

TABLE 15 Examples of GGPP synthase polypeptides. Genbank Row ACCESSIONGenbank GI 1 AAT92871 51013155 2 XP_447025 50289191 3 NP_984623 451903694 XP_390273 46137163 5 XP_404791 49085320 6 XP_368486 39974191 7 Q922366831550 8 AAO85432 29468176 9 XP_572774 58271236 10 XP_502923 5055090111 AAK11525 13021716 12 XP_326920 32412880 13 CAF32032 42820719 14BAD29965 50355599 15 XP_384767 46117498 16 BAD29970 50355631 17 CAB891157649674 18 CAG09545 47229030 19 CAI13753 55960163 20 AAH69913 4712411621 AAH67768 45709211 22 XP_455003 50309979 23 P56966 9296978 24NP_001007 56090562 25 AAT65717 49409613 26 NP_956329 41053321 27BAA90525 6899844 28 XP_405729 49087630 29 AAK11531 13021724 30 XP_41228049119197 31 AAC05273 2944400 32 NP_523958 24660002 33 XP_402074 4907612834 EAL30191 54641441 35 XP_536340 57084951 36 XP_424685 50811194 37AAH06798 13905030 38 AAP06018 29841005 39 XP_460338 50423511 40 AAC055952957271 41 EAK92197 46432727 42 XP_535573 57108760 43 AAH83212 5373459444 XP_486466 51827552 45 CAH18006 51469024 46 CAA75568 3549881 47XP_397455 48143654 48 XP_410947 49101294 49 XP_381914 46109712 50XP_364478 39959279 51 XP_360889 39942704 52 XP_369218 39975655 53XP_406544 49089926 54 XP_367595 39972409 55 XP_363775 39952117 56XP_368486 39974191 57 XP_390273 46137163 58 Q92236 6831550 59 AAK1152513021716 60 CAF32032 42820719 61 XP_404791 49085320 62 AAO85432 2946817663 BAD29965 50355599 64 BAD29970 50355631 65 BAA90525 6899844 66AAT65717 49409613 67 XP_384767 46117498 68 CAB89115 7649674 69 XP_57277458271236 70 AAK11531 13021724 71 XP_502923 50550901 72 CAI13753 5596016373 CAG09545 47229030 74 XP_412280 49119197 75 P56966 9296978 76NP_001007 56090562 77 AAH69913 47124116 78 AAH67768 45709211 79NP_956329 41053321 80 EAL30191 54641441 81 XP_424685 50811194 82XP_536340 57084951 83 NP_523958 24660002 84 AAC05273 2944400 85XP_405729 49087630 86 AAC05595 2957271 87 XP_402074 49076128 88 AAP0601829841005 89 AAH06798 13905030 90 XP_535573 57108760 91 AAH83212 5373459492 AAP21085 30097620 93 NP_984623 45190369 94 XP_447025 50289191 95AAT92871 51013155 96 XP_486466 51827552 97 XP_410947 49101294 98XP_397455 48143654 99 XP_455003 50309979 100 EAK92197 46432727 101XP_381914 46109712 102 XP_460338 50423511 103 CAH18006 51469024 104XP_360889 39942704 105 XP_406544 49089926 106 XP_364478 39959279 107XP_363775 39952117 108 XP_367595 39972409 109 XP_369218 39975655 110C39273 483124 111 BAB79600 18143445 112 BAA14124 216682 113 AAN8559627228290 114 AAA32797 413730 115 Q08291 585326 116 S52584 1073293 117S53722 1076576 118 AAC44848 1842242 119 BAA19583 1944371 120 S712302129674 121 BAA23157 2578822 122 AAC77874 3885426 123 CAB38744 4490594124 BAA78047 4958920 125 BAA82613 5631295 126 CAB56064 5912297 127BAA86284 6277254 128 T11021 7447356 129 AAF78199 8650415 130 AAG104249971808 131 CAC10561 10637876 132 T50879 11279298 133 BAB01343 11994221134 Q42698 13431546 135 Q43133 13431547 136 P54976 13878921 137 BAB5060014023995 138 BAB60678 14325238 139 BAB60820 14422402 140 NP_18958915228704 141 NP_188651 15231055 142 NP_188069 15231869 143 NP_18807315231881 144 AAL01997 15553715 145 AAL01998 15553717 146 NP_25273215599238 147 NP_245470 15602398 148 NP_390308 16079484 149 NP_44001016329282 150 NP_440010 16329282 151 AAL17614 17352451 152 NP_52034317546941 153 AAL76349 18645048 154 AAM21638 20386366 155 AAM2163920386368 156 NP_622916 20807745 157 AAM48650 21328644 158 NP_65979421492720 159 AAM64496 21592547 160 AAM65107 21593158 161 NP_68081122297564 162 ZP_000474 23003800 163 ZP_001252 23469933 164 NP_69876023502633 165 E84566 25313373 166 F85434 25313385 167 AC1245 25313389 168E83997 25313393 169 G84566 25313395 170 AH2910 25315863 171 D8750525398795 172 A89932 25505949 173 F97685 25520741 174 AI3285 25527013 175BAC42571 26450928 176 NP_785195 28378303 177 NP_790546 28867927 178AAO63392 28950937 179 AAO93113 29893480 180 NP_833891 30022260 181AAP59037 31621279 182 ZP_001374 32039216 183 NP_864766 32471772 184NP_875521 33240579 185 NP_881399 33593755 186 NP_884694 33597051 187NP_888456 33600896 188 NP_893187 33861626 189 NP_894940 33863380 190NP_896835 33865276 191 NP_896835 33865276 192 AAQ65086 34365549 193NP_945877 39933601 194 NP_946867 39934591 195 NP_952815 39996864 196AAR37805 40062934 197 AAR37858 40062988 198 AAR98495 41018904 199AAR99082 41059107 200 NP_965349 42519419 201 NP_980544 42783297 202EAA96348 42858148 203 EAB36506 42939031 204 EAB36642 42939300 205EAC39208 43146996 206 EAD26007 43320598 207 EAE43084 43576643 208EAE70061 43630884 209 EAF70308 43832107 210 EAG88494 44055952 211EAH52060 44173220 212 EAH78354 44221788 213 EAH84117 44231960 214EAI11762 44272832 215 EAI49391 44328289 216 EAI54846 44336042 217EAI68356 44355138 218 EAI68713 44355672 219 EAI69401 44356609 220EAI73873 44362658 221 EAJ73634 44506168 222 EAJ77351 44511694 223EAK70639 44644254 224 ZP_001751 45523854 225 AAS76253 45752710 226ZP_001957 45916757 227 1RTRB 46015556 228 ZP_001863 46105954 229ZP_002002 46107045 230 ZP_001711 46132567 231 ZP_002073 46192680 232ZP_002074 46192861 233 AAS82860 46241274 234 ZP_002108 46308696 235YP_010568 46579760 236 BAD18313 47076770 237 ZP_002315 47093750 238ZP_002335 47095946 239 AAT35222 47531118 240 ZP_002401 47569437 241ZP_002435 47573473 242 ZP_002626 48728941 243 ZP_002702 48765678 244ZP_002705 48766028 245 ZP_002732 48768894 246 ZP_002914 48834438 247ZP_003024 48848203 248 ZP_003129 48858958 249 ZP_003177 48863841 250ZP_003225 48869790 251 AAT51323 49086036 252 ZP_003301 49236117 253YP_034222 49476181 254 YP_040995 49483771 255 YP_043579 49486358 256AAT71982 50253560 257 AAT90315 50952782 258 YP_066435 51246551 259YP_075673 51892982 260 YP_085511 52141318 261 YP_092166 52786337 262ZP_001298 53691368 263 YP_105136 53716444 264 YP_111769 53722784 265ZP_003630 54030933 266 YP_129021 54308001 267 AAV74395 56122554 268AAV74396 56122556 269 YP_148246 56420928 270 YP_156518 56461237 271YP_162590 56551751 272 YP_171470 56750769 273 YP_175959 56964228 274YP_186407 57650478 275 YP_190690 58038726 276 AAW66658 58201026 277YP_194187 58337602 278 YP_197469 58579257 279 YP_201938 58582922 280YP_196510 58617311

TABLE 16 Examples of squalene synthase polypeptides. Genbank RowACCESSION Genbank GI 1 AAA34597 171481 2 CAA42583 3686 3 Q9HGZ6 517043364 BAB12207 9955387 5 XP_453457 50306959 6 Q752X9 51701405 7 O7416551701378 8 XP_458579 50420093 9 EAK95451 46436082 10 P78589 2499979 11Q9Y753 51701459 12 XP_407513 49092104 13 XP_364394 39958237 14 Q7S4Z651701416 15 CAD60581 27764301 16 XP_389557 46135731 17 NP_59536319112155 18 B48057 477750 19 NP_034321 34328173 20 CAH92517 55731622 21AAF00038 6002565 22 P53798 1706773 23 NP_004453 31542632 24 AAP3667130584837 25 1EZFC 11514497 26 AAH09251 14328083 27 AAH84016 54035372 28I52090 2136196 29 XP_420043 50745256 30 AAH81810 51858605 31 CAE4836350841455 32 XP_569783 58265254 33 XP_569782 58265252 34 XP_53455757105080 35 XP_401989 49075920

TABLE 17 Examples of phytoene dehydrogenase polypeptides. Genbank RowACCESSION 1 1613414B 2 1613414F 3 1904206A 4 2121278A 5 A86203 6 A966127 A99470 8 AAA24820 9 AAA34001 10 AAA50313 11 AAA64981 12 AAA91161 13AAA99519 14 AAC44798 15 AAC44850 16 AAC48983 17 AAF78201 18 AAG10426 19AAG14399 20 AAG28700 21 AAG50743 22 AAH85048 23 AAK51545 24 AAK51557 25AAK64299 26 AAL02000 27 AAL15300 28 AAL38046 29 AAL73986 30 AAL80005 31AAL91366 32 AAM45380 33 AAM48646 34 AAM63349 35 AAM94364 36 AAN75037 37AAN85599 38 AAO24235 39 AAO46892 40 AAO46894 41 AAO53257 42 AAO53258 43AAO64750 44 AAO93135 45 AAP59036 46 AAP79175 47 AAQ04224 48 AAQ04225 49AAQ65246 50 AAQ65246 51 AAQ88931 52 AAR37797 53 AAR37802 54 AAR37850 55AAR37855 56 AAR86105 57 AAR98491 58 AAR98494 59 AAR98733 60 AAS17750 61AAT01639 62 AAT35222 63 AAT74579 64 AAT74580 65 AAT76050 66 AAT76434 67AAT90316 68 AAU34019 69 AAW23161 70 AB2035 71 AB2064 72 AC2446 73 AF155774 AF2029 75 AG2103 76 AG2509 77 AH1199 78 AI2185 79 AI2273 80 B55548 81B84327 82 B90061 83 BAA14127 84 BAA20276 85 BAA76534 86 BAB10768 87BAB50520 88 BAB51896 89 BAB68552 90 BAB79603 91 BAB82461 92 BAB82462 93BAB98016 94 BAC75676 95 BAC77668 96 BAC77671 97 BAD07279 98 BAD07280 99BAD07287 100 BAD07288 101 CAA52098 102 CAA60479 103 CAA66626 104CAB38739 105 CAB38743 106 CAB40843 107 CAB56041 108 CAB56062 109CAB59726 110 CAB65434 111 CAB94794 112 CAC85667 113 CAD19989 114CAD27442 115 CAD55814 116 CAE00192 117 CAE83576 118 CAF19330 119CAF21094 120 CAF21337 121 CAH91165 122 E90061 123 EAA90383 124 EAA98598125 EAB09790 126 EAB14136 127 EAB18725 128 EAB29729 129 EAB30992 130EAB41377 131 EAB54727 132 EAB76679 133 EAB87028 134 EAB92587 135EAB94459 136 EAB96864 137 EAC01884 138 EAC38895 139 EAC60360 140EAD05874 141 EAD05999 142 EAD20520 143 EAE06978 144 EAE70773 145EAF04985 146 EAF51354 147 EAF62819 148 EAF75453 149 EAG09111 150EAG19412 151 EAG23070 152 EAG25053 153 EAG25054 154 EAG29279 155EAG39845 156 EAG56100 157 EAG63013 158 EAG68633 159 EAG71574 160EAG89835 161 EAH04928 162 EAH04936 163 EAH08586 164 EAH22597 165EAH22853 166 EAH31648 167 EAH55579 168 EAH68071 169 EAH73302 170EAH79041 171 EAH86965 172 EAH97108 173 EAH99977 174 EAI01660 175EAI03576 176 EAI06784 177 EAI11087 178 EAI15261 179 EAI15547 180EAI17521 181 EAI21398 182 EAI29728 183 EAI38468 184 EAI43591 185EAI51589 186 EAI58453 187 EAI72974 188 EAI77885 189 EAI78272 190EAI80262 191 EAI83937 192 EAI86664 193 EAJ00517 194 EAJ05570 195EAJ08238 196 EAJ15524 197 EAJ18144 198 EAJ20649 199 EAJ21683 200EAJ24413 201 EAJ28774 202 EAJ30522 203 EAJ35157 204 EAJ37407 205EAJ39929 206 EAJ54356 207 EAJ54959 208 EAJ56207 209 EAJ58447 210EAJ59958 211 EAJ63347 212 EAJ66054 213 EAJ67637 214 EAJ69812 215EAJ74441 216 EAJ76472 217 EAJ76473 218 EAJ80355 219 EAJ80839 220EAJ81408 221 EAJ86174 222 EAJ87600 223 EAJ88203 224 EAJ88682 225EAJ92341 226 EAJ94774 227 EAJ97555 228 EAJ97958 229 EAK07654 230EAK08513 231 EAK08529 232 EAK10609 233 EAK10614 234 EAK12902 235EAK13034 236 EAK15092 237 EAK22483 238 EAK23222 239 EAK24187 240EAK24674 241 EAK28785 242 EAK34731 243 EAK34742 244 EAK36883 245EAK37522 246 EAK42705 247 EAK43213 248 EAK52580 249 EAK53452 250EAK58759 251 EAK62665 252 EAK63558 253 F84187 254 F90272 255 G87635 256G90413 257 H83880 258 H84320 259 JC7723 260 NP_060220 261 NP_080435 262NP_193157 263 NP_214383 264 NP_276913 265 NP_293819 266 NP_294534 267NP_294585 268 NP_295972 269 NP_338490 270 NP_376437 271 NP_377056 272NP_388895 273 NP_441167 274 NP_441254 275 NP_442491 276 NP_442727 277NP_562475 278 NP_568712 279 NP_601630 280 NP_601630 281 NP_616426 282NP_624522 283 NP_626360 284 NP_630834 285 NP_643053 286 NP_647302 287NP_659552 288 NP_661086 289 NP_661546 290 NP_661701 291 NP_662300 292NP_681023 293 NP_681127 294 NP_682351 295 NP_693380 296 NP_693382 297NP_737250 298 NP_763380 299 NP_786524 300 NP_822198 301 NP_822828 302NP_827278 303 NP_851528 304 NP_857496 305 NP_868798 306 NP_869339 307NP_870237 308 NP_874530 309 NP_874561 310 NP_874977 311 NP_892236 312NP_892265 313 NP_892458 314 NP_893232 315 NP_894882 316 NP_895385 317NP_895793 318 NP_895829 319 NP_896854 320 NP_896994 321 NP_898304 322NP_898346 323 NP_902647 324 NP_923340 325 NP_923639 326 NP_923813 327NP_925079 328 NP_931515 329 NP_936379 330 NP_940208 331 NP_945754 332NP_946860 333 NP_946866 334 NP_948851 335 NP_962004 336 NP_968600 337NP_974222 338 NP_974545 339 O49901 340 P17059 341 P54971 342 P54978 343P54979 344 P54981 345 P54982 346 P74306 347 Q01671 348 Q02861 349 Q38893350 Q40406 351 Q9FV46 352 Q9SE20 353 Q9SMJ3 354 Q9ZTN9 355 Q9ZTP4 356S29314 357 S32171 358 S49624 359 S52586 360 S65060 361 T10701 362 T31463363 T46822 364 T48646 365 T50745 366 T50749 367 T50893 368 T50910 369T51119 370 T51123 371 XP_324732 372 XP_383241 373 XP_401825 374XP_470568 375 XP_473486 376 XP_477063 377 XP_525801 378 XP_540198 379YP_006049 380 YP_013621 381 YP_024310 382 YP_041986 383 YP_041988 384YP_044561 385 YP_044564 386 YP_062471 387 YP_117947 388 YP_120612 389YP_135077 390 YP_136483 391 YP_145331 392 YP_145348 393 YP_171014 394YP_172823 395 YP_173078 396 YP_173207 397 YP_184572 398 YP_187368 399YP_187371 400 YP_187371 401 YP_187371 402 ZP_000490 403 ZP_000509 404ZP_000518 405 ZP_000566 406 ZP_000627 407 ZP_000627 408 ZP_001073 409ZP_001081 410 ZP_001091 411 ZP_001116 412 ZP_001117 413 ZP_001119 414ZP_001124 415 ZP_001510 416 ZP_001591 417 ZP_001593 418 ZP_001602 419ZP_001614 420 ZP_001645 421 ZP_001650 422 ZP_001722 423 ZP_001746 424ZP_001752 425 ZP_001770 426 ZP_001777 427 ZP_001787 428 ZP_001837 429ZP_001867 430 ZP_002073 431 ZP_002077 432 ZP_002339 433 ZP_002680 434ZP_002705 435 ZP_002771 436 ZP_002892 437 ZP_002916 438 ZP_002963 439ZP_003022 440 ZP_003036 441 ZP_003107 442 ZP_003202 443 ZP_003258 444ZP_003268 445 ZP_003269 446 ZP_003276 447 ZP_003283 448 ZP_003557 449ZP_003559 450 ZP_003565 451 ZP_003577 452 ZP_003593 453 ZP_003595 441ZP_003685

TABLE 18 Examples of phytoene synthase and lycopene cyclasepolypeptides. Genbank Row Accession Genbank GI 1 1613414C 227040 2A49558 1076590 3 AAA19428 506623 4 AAA32836 413732 5 AAA64982 148413 6AAB87738 29893495 7 AAC44849 1842243 8 AAD38051 13542332 9 AAF782028650418 10 AAF82616 9081847 11 AAG10427 9971814 12 AAG28701 11066678 13AAK07734 18476085 14 AAK07735 18476089 15 AAK15621 13195243 16 AAL0200115553721 17 AAL76346 18645045 18 AAL82578 21326700 19 AAM45379 2136035320 AAM48647 21328641 21 AAM62787 21553694 22 AAM94363 22296799 23AAN85600 27228294 24 AAO24767 27903500 25 AAO39835 28403302 26 AAO4689537729028 27 AAO47570 33465823 28 AAO73816 33465821 29 AAP22038 3034941430 AAP55451 32350232 31 AAP55453 32350236 32 AAP55461 32350252 33AAP55471 32350272 34 AAP55484 32350298 35 AAP55486 32350302 36 AAP5608332349564 37 AAP56124 32349646 38 AAP56127 32349652 39 AAP56136 3234967040 AAP56148 32349694 41 AAP56155 32349708 42 AAP56156 32349710 43AAP56157 32349712 44 AAP56158 32349714 45 AAP79176 32307542 46 AAQ9183737499616 47 AAR08445 38037628 48 AAR31885 39842609 49 AAR37803 4006293250 AAR37856 40062986 51 AAR86104 40456029 52 AAR87868 40557193 53AAR98492 41018901 54 AAS02284 41394357 55 AAS17009 42491736 56 AAS1830742521626 57 AAT28184 47498515 58 AAT35222 47531118 59 AAT38473 4777918160 AAT46069 48686711 61 AAT74581 50313418 62 AAT90319 50952786 63AAV74394 56122551 64 AAW23162 56698928 65 AC2035 25366683 66 AC203525366683 67 BAB18514 11344507 68 BAB79604 18143449 69 BAD07278 4080973970 BAD07286 40809755 71 BAD62106 54291340 72 BAD62107 54291341 73 C9006125506636 74 CAA47625 19347 75 CAA68575 19341 76 CAB07958 1934837 77CAB38740 4490590 78 CAB51949 5690074 79 CAB56063 5912296 80 CAB863887453011 81 CAB93661 8250520 82 CAB94795 8574392 83 CAC19567 11990226 84CAC27383 12584564 85 CAD19988 18307500 86 CAD29284 57282088 87 CAE7660938567321 88 E37802 95606 89 E84320 25410251 90 EAA98758 42863045 91EAB01965 42869439 92 EAB04170 42873822 93 EAB07138 42879858 94 EAB0979142885235 95 EAB19826 42905452 96 EAB35029 42936011 97 EAB41375 4294874098 EAB78706 43024004 99 EAB92586 43052355 100 EAC06949 43081493 101EAC18360 43104624 102 EAC25793 43119723 103 EAC29883 43128092 104EAC32813 43133973 105 EAC33105 43134560 106 EAC38486 43145552 107EAC52233 43173313 108 EAC60029 43189028 109 EAC68026 43204953 110EAC96197 43261031 111 EAD08701 43285745 112 EAD20866 43310220 113EAD32755 43334458 114 EAD38008 43345761 115 EAD50152 43370658 116EAD50402 43371147 117 EAD81123 43452903 118 EAD93882 43478303 119EAE12860 43516265 120 EAE16121 43522884 121 EAE31084 43552634 122EAE35665 43561764 123 EAE44717 43579862 124 EAE46627 43583580 125EAE47846 43586023 126 EAE72264 43635190 127 EAE76009 43642573 128EAE86335 43662748 129 EAE89581 43669163 130 EAF18881 43728007 131EAF64277 43819669 132 EAF67931 43827263 133 EAF84745 43861327 134EAF94004 43880040 135 EAG06083 43903395 136 EAG21950 43933540 137EAG43625 43973477 138 EAG50171 43985555 139 EAG57517 43999205 140EAG62787 44009110 141 EAG65580 44014171 142 EAG68110 44018715 143EAG72283 44026322 144 EAG78750 44037938 145 EAG80445 44041116 146EAG93220 44064453 147 EAH04927 44085694 148 EAH08972 44093217 149EAH10377 44095788 150 EAH22151 44117864 151 EAH31461 44134654 152EAH50033 44169323 153 EAH64480 44196848 154 EAH79040 44223009 155EAH99976 44255671 156 EAI02786 44259828 157 EAI02787 44259829 158EAI03575 44260943 159 EAI05900 44264266 160 EAI61004 44344824 161EAI70669 44358327 162 EAI83938 44377067 163 EAJ05110 44406802 164EAJ05569 44407471 165 EAJ08876 44412338 166 EAJ35156 44449986 167EAJ38900 44455130 168 EAJ49645 44470504 169 EAJ54357 44477026 170EAJ60475 44485647 171 EAJ64125 44492007 172 EAJ67499 44497025 173EAJ76471 44510405 174 EAJ76950 44511114 175 EAJ78637 44513596 176EAJ78787 44513824 177 EAJ79616 44515082 178 EAJ80356 44516200 179EAJ81914 44518489 180 EAJ87417 44526623 181 EAK08514 44557109 182EAK08523 44557119 183 EAK12901 44563097 184 EAK22180 44576315 185EAK24859 44580262 186 EAK28345 44585276 187 EAK34732 44594324 188EAK34736 44594329 189 EAK37296 44597942 190 EAK37521 44598256 191EAK56335 44624430 192 G84363 25410528 193 NP_274195 15677043 194NP_284085 15794263 195 NP_294586 15805888 196 NP_388961 16078144 197NP_441168 16330440 198 NP_443763 16519643 199 NP_624523 21218744 200NP_630832 21225053 201 NP_662273 21674208 202 NP_682350 22299103 203NP_693381 23099915 204 NP_786525 28379633 205 NP_822199 29827565 206NP_822829 29828195 207 NP_851527 30795077 208 NP_868799 32475805 209NP_874560 33239618 210 NP_879992 33592348 211 NP_884101 33596458 212NP_889809 33602249 213 NP_892264 33860703 214 NP_895828 33864268 215NP_898345 33866786 216 NP_902648 34498433 217 NP_902649 34498434 218NP_924690 37521313 219 NP_931516 37528171 220 NP_946861 39934585 221NP_949079 39936803 222 NP_962005 41409169 223 NP_968601 42523221 224O07333 3913360 225 P08196 585746 226 P21683 30923192 227 P37269 585009228 P37271 27735222 229 P37272 585749 230 P53797 1709885 231 P549751706137 232 P54977 1706139 233 P65860 54041032 234 Q9SSU8 8928282 235Q9UUQ6 34922667 236 S22474 7489041 237 S32170 321671 238 S52587 1073300239 S56668 2129505 240 S68307 2130144 241 T10702 7484346 242 T4659411291807 243 T50746 11356347 244 T50895 11291816 245 XP_324765 32408567246 XP_383242 46114448 247 XP_403902 49080862 248 YP_006040 46255128 249YP_103126 53723680 250 YP_112342 53723357 251 YP_117945 54023703 252YP_120611 54026369 253 YP_136628 55378778 254 YP_136629 55378779 255YP_145340 55978284 256 YP_145343 55978287 257 YP_160917 56479328 258YP_160918 56479329 259 YP_162605 56551766 260 YP_172822 56752121 261YP_187369 57652299 262 YP_192648 58040684 263 ZP_000044 22956752 264ZP_001091 53688068 265 ZP_001591 53763709 266 ZP_001657 45514234 267ZP_001690 46132223 268 ZP_001746 45523280 269 ZP_001837 53771530 270ZP_001867 45546711 271 ZP_002096 46204978 272 ZP_002248 46324513 273ZP_002450 47575031 274 ZP_002680 48763469 275 ZP_002710 48766450 276ZP_002791 48782680 277 ZP_002892 48832182 278 ZP_002916 48834623 279ZP_003036 48849426 280 ZP_003269 48893702 281 ZP_003351 52007802 282ZP_003487 53730362 283 ZP_003501 53759405 284 ZP_003591 53798896 285ZP_003628 54030691

TABLE 19 Examples of carotenoid ketolase polypeptides. Accession RowNumber GI Number 1 AAA99932 609575 2 AAB48668 1870215 3 AAC25611 25419364 AAF78203 8650419 5 AAH16427 16741158 6 AAN03484 22597194 7 AAN8549726541510 8 AAN86030 33439708 9 AAO64399 28976134 10 AAQ23139 33621091 11AAT35222 47531118 12 AAT35555 47558911 13 AAV41372 55139370 14 AB230725530134 15 AF2204 25533132 16 BAB54999 14028447 17 BAB58879 14270087 18BAC98366 37360914 19 CAA60478 2654318 20 CAB56059 5912292 21 D8767325398945 22 EAA79304 42823978 23 EAA80363 42826055 24 EAA81403 4282811525 EAA84711 42834481 26 EAB82380 43031476 27 EAB86624 43040184 28EAC05755 43079085 29 EAD12219 43292778 30 EAD71182 43427899 31 EAD9492743480380 32 EAF11582 43712986 33 EAF98072 43888329 34 EAG19345 4392873835 EAG38273 43963688 36 EAG79800 44039853 37 EAG96474 44070318 38EAH00349 44077315 39 EAH36448 44143633 40 EAH40683 44151265 41 EAH5318044175316 42 EAH96648 44250729 43 EAI05260 44263397 44 EAI17468 4428132945 EAI53009 44333409 46 EAI54054 44334878 47 EAI67818 44354362 48EAI68153 44354875 49 EAI89684 44384943 50 EAJ27674 44439188 51 EAJ4558944464684 52 EAJ45589 44464684 53 EAJ67118 44496466 54 EAJ74221 4450702255 EAJ74653 44507662 56 EAJ88396 44528064 57 EAJ88887 44528792 58EAK06069 44553531 59 EAK11467 44561166 60 EAK16824 44568733 61 EAK2882844585942 62 EAK28828 44585942 63 EAK31112 44589271 64 EAK42591 4460544165 NP_045063 11465545 66 NP_081575 27754029 67 NP_338204 15843167 68NP_440788 16330060 69 NP_441220 16330492 70 NP_682690 22299443 71NP_770721 27379192 72 NP_848964 30468077 73 NP_857223 31794730 74NP_881760 33594116 75 NP_882469 33594826 76 NP_886657 33599097 77NP_895643 33864083 78 NP_896386 33864827 79 NP_897461 33865902 80NP_924674 37521297 81 NP_927525 37524181 82 NP_947075 39934799 83 P549721706150 84 Q39982 2498257 85 Q44261 2498256 86 T31123 11361063 87XP_330780 32420673 88 XP_368852 39974923 89 XP_380194 46102628 90XP_383758 46115480 91 XP_405100 49086048 92 XP_409222 49095522 93YP_102417 53725671 94 YP_108945 53719959 95 YP_132414 54302421 96YP_154670 56459389 97 YP_166682 56696325 98 YP_168846 56698471 99YP_172377 56751676 100 ZP_001068 23124870 101 ZP_001112 53688676 102ZP_001607 53764743 103 ZP_001757 46118877 104 ZP_001787 53736018 105ZP_002218 46321435 106 ZP_002456 47575608 107 ZP_003028 48848557 108ZP_003107 48856640 109 ZP_003264 48893204 110 ZP_003458 53688805 111ZP_003513 53763576

TABLE 20 Examples of carotenoid hydroxylase polypeptides. Genbank RowACCESSION Genbank GI 1 AAC44852 1842246 2 AAC49443 1575296 3 AAD542435852870 4 AAG10430 9971820 5 AAG10793 9988836 6 AAG33636 11245486 7AAL80006 19071768 8 AAM44971 21280903 9 AAM51300 21436107 10 AAM7700721734857 11 AAN85601 27228295 12 AAO53295 28911949 13 AAS48097 4488764214 AAS55552 45184599 15 AAS88426 46326968 16 AAT48741 49036137 17AAT84408 50844570 18 AAV85452 56267980 19 AAV85453 56267982 20 BAA14129216687 21 BAB79605 18143450 22 BAC77670 31790567 23 BAD07283 40809749 24BAD07291 40809765 25 CAA70427 2956671 26 CAA70888 2956717 27 CAB556255870598 28 CAB55626 5870600 29 CAB56060 5912293 30 CAC06712 9968545 31CAC95130 33145986 32 EAB30128 42926157 33 EAC49462 43167766 34 EAC8612943241003 35 EAD61089 43395962 36 EAD76156 43443111 37 EAD88640 4346779338 EAE27903 43546376 39 EAE28203 43546980 40 EAE78743 43647896 41EAF12173 43714211 42 EAH29370 44130906 43 EAH44202 44158360 44 EAI0076644256844 45 EAI29017 44298625 46 EAJ30844 44443849 47 EAJ72524 4450451648 EAK10611 44559981 49 EAK53455 44620561 50 EAK63955 44635271 51 H9046925394049 52 NP_745389 26989964 53 NP_922503 37536402 54 P54973 170615255 Q44262 2498258 56 S52982 1073291 57 XP_473611 50928167 58 YP_02430948478603 59 ZP_003055 48851297 60 ZP_003107 48856620

TABLE 21 Examples of astaxanthin synthase polypeptides and putativeastaxanthin synthase polypeptides. Genbank Row ACCESSION Genbank GI 1AAM56288 21501451 2 XP_571276 58268240 3 EAL20013 50257304 4 XP_40180449075484 5 XP_397817 49067054 6 XP_399595 49070612 7 XP_403279 490792188 XP_382294 46110473 9 XP_406021 49088382 10 XP_381224 46108332 11XP_391479 46139577 12 XP_569261 58264210 13 EAL22841 50260180 14XP_359866 39940658

TABLE 22 Examples of carotenoid epsilon hydroxylase polypeptides.ACCESSION GI PROTEIN DESCRIPTION ABB52076 79155148 putative epsilon-ringcarotene hydroxylase [Daucus carota subsp. sativus] BAD94136 62319017Cytochrom P450-like protein [Arabidopsis thaliana] ABD28565 87162770E-class P450, group I [Medicago truncatula] AAT28222 47498772 putative97B2-like cytochrome P450 [Ginkgo biloba] ABC68396 85001685 cytochromeP450 monooxygenase CYP97A [Glycine max] ABC59110 84514203 cytochromeP450 monooxygenase CYP97B [Medicago truncatula] NP_190881 42565881 LUT1(LUTEIN DEFICIENT 1); oxygen binding [Arabidopsis thaliana] ABB4795478708979 cytochrome P450 monooxygenase, putative [Oryza sativa (japonicacultivar-group)] NP_922604 37536604 putative cytochrome P450monooxygenase [Oryza sativa (japonica cultivar-group)]

TABLE 23 Examples of lycopene cyclase polypeptides, beta and epsilonsubunits. ACCESSION GI PROTEIN DESCRIPTION AAK07431 12746307 lycopeneepsilon-cyclase [Adonis palaestina] ABB52073 79154988 putative lycopeneepsilon cyclase [Daucus carota subsp. sativus] Q38932 27735211 Lycopeneepsilon cyclase, chloroplast precursor AAB53336 1399181 lycopene epsiloncyclase AAG10428 9971816 epsilon cyclase [Tagetes erecta] AAK0743412746313 lycopene epsilon-cyclase [Lactuca sativa] AAM45382 21360359epsilon cyclase [Tagetes erecta] O65837 11132841 Lycopene epsiloncyclase, chloroplast precursor AAL69394 18419661 lycopeneepsilon-cyclase [Spinacia oleracea] BAE79549 87299433 lycopeneepsilon-cyclase [Chrysanthemum x morifolium] XP_463351 50901836 putativelycopene epsilon-cyclase [Oryza sativa (japonica cultivar-group)]AAS48096 44887640 epsilon lycopene cyclase [Citrus sinensis] AAX9267962638188 lycopene epsilon cyclase [Citrus maxima] AAL92114 19569601lycopene epsilon-cyclase [Citrus x paradisi] AAK07433 12746311 lycopeneepsilon-cyclase [Solanum tuberosum] AAL47019 17864021 lycopeneepsilon-cyclase [Citrus sinensis] AAT46065 48686703 chloroplast lycopeneepsilon-cyclase precursor [Chlamydomonas reinhardtii] BAD07293 40809769lycopene epsilon-cyclase [Citrus limon] BAD07285 40809753 lycopeneepsilon-cyclase [Citrus sinensis] BAD07277 40809737 lycopeneepsilon-cyclase [Citrus unshiu] EAJ62839 44489138 unknown [environmentalsequence] BAE43547 73993068 putative lycopene beta cyclase [Taxodiumdistichum var. distichum] BAE43550 73993074 putative lycopene betacyclase [Taxodium distichum var. distichum] BAE43557 73993088 putativelycopene beta cyclase [Taxodium distichum var. imbricarium] BAE4355873993090 putative lycopene beta cyclase [Taxodium distichum var.imbricarium] BAE43553 73993080 putative lycopene beta cyclase [Taxodiumdistichum var. imbricarium] BAE43545 73993064 putative lycopene betacyclase [Taxodium distichum var. distichum] BAE43556 73993086 putativelycopene beta cyclase [Taxodium distichum var. imbricarium] BAE4355273993078 putative lycopene beta cyclase [Taxodium distichum var.distichum] BAE43560 73993094 putative lycopene beta cyclase [Taxodiumdistichum var. imbricarium] BAE43554 73993082 putative lycopene betacyclase [Taxodium distichum var. imbricarium] BAE43551 73993076 putativelycopene beta cyclase [Taxodium distichum var. distichum] BAE4351973993012 putative lycopene beta cyclase [Cryptomeria japonica] BAE4353573993044 putative lycopene beta cyclase [Cryptomeria japonica] BAE4354173993056 putative lycopene beta cyclase [Cryptomeria japonica] BAE4354273993058 putative lycopene beta cyclase [Cryptomeria japonica] BAE4351773993008 putative lycopene beta cyclase [Cryptomeria japonica] BAE4353473993042 putative lycopene beta cyclase [Cryptomeria japonica] BAE4353773993048 putative lycopene beta cyclase [Cryptomeria japonica] BAE4353373993040 putative lycopene beta cyclase [Cryptomeria japonica] BAD0277438603277 putative lycopene beta cyclase [Cryptomeria japonica] BAD0276638603261 putative lycopene beta cyclase [Cryptomeria japonica] BAE4354073993054 putative lycopene beta cyclase [Cryptomeria japonica] BAE4351473993002 putative lycopene beta cyclase [Cryptomeria japonica] BAE4354473993062 putative lycopene beta cyclase [Cryptomeria japonica] BAE4353873993050 putative lycopene beta cyclase [Cryptomeria japonica] BAE4352873993030 putative lycopene beta cyclase [Cryptomeria japonica] BAE4354673993066 putative lycopene beta cyclase [Taxodium distichum var.distichum] BAE43526 73993026 putative lycopene beta cyclase [Cryptomeriajaponica] BAE43543 73993060 putative lycopene beta cyclase [Cryptomeriajaponica] BAD02742 38603213 putative lycopene beta cyclase [Cryptomeriajaponica] BAD02770 38603269 putative lycopene beta cyclase [Cryptomeriajaponica] BAE43522 73993018 putative lycopene beta cyclase [Cryptomeriajaponica] BAE43559 73993092 putative lycopene beta cyclase [Taxodiumdistichum var. imbricarium] BAE43527 73993028 putative lycopene betacyclase [Cryptomeria japonica] BAE43548 73993070 putative lycopene betacyclase [Taxodium distichum var. distichum] AAF44700 14550425 lycopenebeta-cyclase [Citrus sinensis] BAE43555 73993084 putative lycopene betacyclase [Taxodium distichum var. imbricarium] BAE43549 73993072 putativelycopene beta cyclase [Taxodium distichum var. distichum] AAU1414451922063 lycopene beta-cyclase [Citrus sinensis] AAN86060 27261727lycopene cyclase [Citrus unshiu] AAR89632 40756518 lycopene-beta-cyclase[Citrus maxima] AAM21152 20530862 lycopene beta-cyclase [Citrussinensis] AAD38049 13959731 lycopene cyclase [Citrus x paradisi]AAU05146 51511939 lycopene beta-cyclase [Citrus sinensis] AAU0514551511937 lycopene beta-cyclase [Citrus sinensis] AAK07430 12746305lycopene beta-cyclase [Adonis palaestina] ABB72443 82394885 lycopenebeta-cyclase [Citrus sinensis] BAE79544 87299423 lycopene beta-cyclase[Chrysanthemum x morifolium] BAE78471 85717882 lycopene beta cyclase[Taraxacum officinale] Q43415 11133019 Lycopene beta cyclase,chloroplast precursor AAF23013 6665782 lycopene epsilon-cyclase [Daucuscarota] ABB52071 79154899 putative lycopene beta cyclase [Daucus carotasubsp. sativus] AAW88382 59665024 lycopene beta-cyclase [Lyciumbarbarum] AAG10429 9971818 beta cyclase [Tagetes erecta] AAM4538121360357 beta cyclase [Tagetes erecta] AAM14335 20259239 putativelycopene beta cyclase [Arabidopsis thaliana] AAO18661 27728515 lycopenebeta-cyclase [Zea mays] AAA81880 735882 lycopene cyclase Q43503 11133022Lycopene beta cyclase, chloroplast precursor S66350 2129931 lycopenebeta-cyclase (EC 5.5.1.—) - tomato XP_464409 50905841 putative lycopenebeta-cyclase [Oryza sativa (japonica cultivar-group)] CAD70565 45237491lycopene cyclase [Bixa orellana] Q43578 11133025 Lycopene beta cyclase,chloroplast precursor AAL92175 19569782 beta-lycopene cyclase[Sandersonia aurantiaca] AAX54906 61742130 putative chloroplast lycopenebeta cyclase precursor [Chlamydomonas reinhardtii] S66349 2129954lycopene beta-cyclase (EC 5.5.1.—) - common tobacco AAG21133 10644119chromoplast-specific lycopene beta-cyclase [Lycopersicon esculentum]CAB92977 8247354 neoxanthin synthase [Solanum tuberosum] CAB933428249885 neoxanthin synthase [Lycopersicon esculentum] Q9SEA0 11131528Capsanthin/capsorubin synthase, chloroplast precursor Q42435 12643508Capsanthin/capsorubin synthase, chloroplast precursor AAO64977 37730608lycopene beta cyclase [Haematococcus pluvialis] Q40424 11133011 Lycopenebeta cyclase, chloroplast precursor ABB52072 79154940 putativecapsanthin-capsorubin synthase [Daucus carota subsp. sativus] AAQ0266833304511 lycopene cyclase [Setaria italica] CAA54961 840729 putativechromoplastic oxydo-reductase [Capsicum annuum] EAJ62838 44489136unknown [environmental sequence] YP_401079 81300871 Lycopene cyclase,beta and epsilon [Synechococcus elongatus PCC 7942] YP_172741 56752040lycopene cyclase [Synechococcus elongatus PCC 6301] ZP_011 . . .88808972 lycopene beta cyclase [Synechococcus sp. WH 7805] EAK5005244615956 unknown [environmental sequence] NP_892751 33861190 putativelycopene epsilon cyclase [Prochlorococcus marinus subsp. pastoris str.CCMP1986] NP_875182 33240240 Lycopene epsilon cyclase [Prochlorococcusmarinus subsp. marinus str. CCMP1375] YP_382237 78213458 Lycopenecyclase, beta and epsilon [Synechococcus sp. CC9605] YP_397130 78779018Lycopene cyclase, beta and epsilon [Prochlorococcus marinus str. MIT9312] NP_896821 33865262 lycopene beta cyclase [Synechococcus sp. WH8102] YP_397570 78779458 Lycopene cyclase, beta and epsilon[Prochlorococcus marinus str. MIT 9312] ZP_010 . . . 87302144 lycopenecyclase [Synechococcus sp. WH 5701] EAK17149 44569190 unknown[environmental sequence] YP_291882 72382527 lycopene cyclase, beta andepsilon [Prochlorococcus marinus str. NATL2A] NP_875528 33240586Lycopene beta cyclase related dehydrogenase [Prochlorococcus marinussubsp. marinus str. CCMP1375] NP_893181 33861620 putative lycopene betacyclase [Prochlorococcus marinus subsp. pastoris str. CCMP1986]NP_895600 33864040 putative lycopene epsilon cyclase [Prochlorococcusmarinus str. MIT 9313] EAI47456 44325573 unknown [environmentalsequence] YP_291268 72381913 lycopene cyclase, beta and epsilon[Prochlorococcus marinus str. NATL2A] ZP_010 . . . 84517806 Lycopenebeta cyclase related dehydrogenase [Prochlorococcus marinus str. MIT9211] AAF34191 6970079 lycopene epsilon cyclase [Daucus carota] ZP_010 .. . 84518202 Lycopene epsilon cyclase [Prochlorococcus marinus str. MIT9211] YP_376736 78184301 Lycopene cyclase, beta and epsilon[Synechococcus sp. CC9902] ZP_003 . . . 66796756 Lycopene cyclase, betaand epsilon [Deinococcus geothermalis DSM 11300] NP_894954 33863394putative lycopene beta cyclase [Prochlorococcus marinus str. MIT 9313]AAT76051 50365502 lycopene cyclase [Citrus clementina] EAK22047 44576122unknown [environmental sequence] NP_294525 15805827 lycopene cyclase[Deinococcus radiodurans R1]

TABLE 24 Examples of carotenoid glucosyltransferase polypeptides.ACCESSION GI PROTEIN DESCRIPTION AAA21261 148395 CrtX [Pantoeaagglomerans] AAN85597 27228291 Zeaxanthin Glucosyl Transferase [Pantoeastewartii] BAB79601 18143446 crtX [Pantoea agglomerans pv. milletiae]AAZ73147 72536082 zeaxanthin glucosyl transferase [Enterobacteriaceaebacterium DC413] AAZ73128 72536060 zeaxanthin glucosyl transferase[Enterobacteriaceae bacterium DC260] AAZ73140 72536074 zeaxanthinglucosyl transferase [Enterobacteriaceae bacterium DC416] Q01330 231911Zeaxanthin glucosyl transferase ZP_006... 71674312UDP-glycosyltransferase, MGT [Trichodesmium erythraeum IMS101] NP_43997216329244 zeaxanthin glucosyl transferase [Synechocystis sp. PCC 6803]EAH29368 44130903 unknown [environmental sequence] ZP_005... 67926135zeaxanthin glucosyl transferase, hypothetical protein [Crocosphaerawatsonii WH 8501] YP_378763 78188425 hypothetical protein Cag_0447[Chlorobium chlorochromatii CaD3] ZP_005... 68549418 Glycosyltransferase, group 1 [Pelodictyon phaeoclathratiforme BU-1] ZP_010...85713606 glycosyl transferase, group 1 [Nitrobacter sp. Nb-311A]YP_317171 75674750 glycosyl transferase, group 1 [Nitrobacterwinogradskyi Nb- 255] ZP_006... 69929171 Glycosyl transferase, group 1[Nitrobacter hamburgensis X14] ZP_009... 84500589 hypothetical proteinOB2597_11541 [Oceanicola batsensis HTCC2597] ZP_009... 83953176hypothetical protein NAS141_12746 [Sulfitobacter sp. NAS- 14.1]ZP_009... 83942121 hypothetical protein EE36_07793 [Sulfitobacter sp.EE-36] YP_508020 89052569 glycosyl transferase, group 1 [Jannaschia sp.CCS1] ZP_010... 85704103 hypothetical protein ROS217_13931 [Roseovariussp. 217] ZP_009... 83370850 probable glycosyltransferase [Rhodobactersphaeroides ATCC 17025] ZP_006... 69934465 Glycosyl transferase, group 1[Paracoccus denitrificans PD1222] ZP_009... 83949880 probableglycosyltransferase [Roseovarius nubinhibens ISM] YP_376237 78183803putative glycosyltransferase [Synechococcus sp. CC9902] YP_37612978183695 probable glycosyltransferase [Synechococcus sp. CC9902]YP_374296 78186253 hypothetical protein Plut_0365 [Pelodictyon luteolumDSM 273] ZP_010... 87301651 Putative glycosyltransferase [Synechococcussp. WH 5701] ZP_011... 88809938 Putative glycosyltransferase[Synechococcus sp. WH 7805] BAE47471 78483937 carotenoidglucosyltransferase [Paracoccus sp. N81106] ZP_010... 87303273 probableglycosyltransferase [Synechococcus sp. WH 5701] YP_376127 78183693probable glycosyltransferase [Synechococcus sp. CC9902] YP_50133488196509 hypothetical protein SAOUHSC_02880 [Staphylococcus aureussubsp. aureus NCTC 8325] YP_187370 57652300 glycosyl transferase, group2 family protein [Staphylococcus aureus subsp. aureus COL] CAA666271340131u nnamed protein product [Staphylococcus aureus] YP_04198749484763 putative glycosyl transferase [Staphylococcus aureus subsp.aureus MRSA252] YP_417885 82752144 hypothetical protein SAB2436c[Staphylococcus aureus RF122] YP_252404 70725490 hypothetical proteinSH0489 [Staphylococcus haemolyticus JCSC1435] NP_693379 23099913hypothetical protein OB2458 [Oceanobacillus iheyensis HTE831] ZP_008...82501285 conserved hypothetical protein [Caldicellulosiruptorsaccharolyticus DSM 8903] ZP_010... 87303565 hypothetical proteinWH5701_09900 [Synechococcus sp. WH 5701]

TABLE 25 Examples of acyl CoA:diacyglycerol acyltransferase (DGAT)polypeptides. ACCESSION GI PROTEIN DESCRIPTION XP_957022 85082953hypothetical protein [Neurospora crassa N150] XP_386864 46124621hypothetical protein FG06688.1 [Gibberella zeae PH-1] XP_755172 71000982diacylglycerol O-acyltransferase DGAT [Aspergillus fumigatus Af293]XP_663763 67539978 hypothetical protein AN6159.2 [Aspergillus nidulansFGSC A4] BAE65302 83775179 unnamed protein product [Aspergillus oryzae]XP_502557 50550169 hypothetical protein [Yarrowia lipolytica] AAS7866256199782 diacylglycerol acyltransferase [Glycine max] ABB84383 82582915diacylglycerol acyltransferase [Jatropha curcas] AAV31083 541454591,2-diacyl-sn-glycerol:acyl-CoA acyltransferase [Euonymus alatus]AAG23696 10803053 diacylglycerol acyltransferase [Perilla frutescens]AAF64065 7576941 putative diacylglycerol acyltransferase [Brassicanapus] AAS01606 41387497 acyl-CoA:diacylglycerol acyltransferase 1 [Oleaeuropaea] AAT73629 50299542 acyl CoA:diacylglycerol acyltransferase[Glycine max] AAM03340 67043496 putative diacylglycerol acyltransferase[Tropaeolum majus] XP_645633 66824557 hypothetical protein DDB0202877[Dictyostelium discoideum] AAF19345 6625653 diacylglycerol acylCoAacyltransferase [Nicotiana tabacum] AAY40785 63376239 diacylglycerolacyltransferase DGAT2 [Brassica juncea] AAW47581 57231736 diacylglycerolacyltransferase [Oryza sativa (japonica cultivar-group)] AAR1147938146080 diacylglycerol acyltransferase [Ricinus communis] AAY4078463376226 diacylglycerol acyltransferase DGAT1 [Brassica juncea] AAP6832231711932 At2g19450 [Arabidopsis thaliana] AAW51456 57545061diacylglycerol acyltransferase [Lotus corniculatus var. japonicus]AAD45536 5579408 putative diacylglycerol acyltransferase [Brassicanapus] BAD53762 53791817 putative acyl-CoA:diacylglycerolacyltransferase [Oryza sativa (japonica cultivar-group)] NP_95602441054343 hypothetical protein LOC325875 [Danio rerio] AAL49962 18642598diacylglycerol acyltransferase 1 [Bos taurus] XP_930884 89028385 similarto Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase)(ACAT-related gene) [Homo sapiens] NP_777118 27819636 diacylglycerolO-acyltransferase 1 [Bos taurus] Q9GMF1 18202926 DiacylglycerolO-acyltransferase 1 (Diglyceride acyltransferase) NP_036211 6912332diacylglycerol O-acyltransferase 1 [Homo sapiens] AAH06263 34782946DGAT1 protein [Homo sapiens] XP_780515 72006039 similar toDiacylglycerol O-acyltransferase 1 [Strongylocentrotus purpuratus]AAD40881 5225382 putative diacylglycerol acyltransferase [Brassicanapus] XP_539214 73974769 similar to Diacylglycerol O-acyltransferase 1(ACAT related gene product 1) isoform 1 [Canis familiaris] AAZ2240371063860 diacylglycerol O-acyltransferase 1 [Bubalus bubalis] NP_99921647522918 diacylglycerol acyltransferase [Sus scrofa] NP_001... 50539976hypothetical protein LOC436731 [Danio rerio] XP_849176 73974767 similarto Diacylglycerol O-acyltransferase 1 (ACAT related gene product 1)isoform 2 [Canis familiaris] NP_505828 71997360 H19N07.4 [Caenorhabditiselegans] AAF82410 9049538 diacylglycerol acyltransferase [Caenorhabditiselegans] CAE75170 39591950 Hypothetical protein CBG23107 [Caenorhabditisbriggsae] XP_626337 66358318 diacylglycerol acyltransferase 1[Cryptosporidium parvum Iowa II] XP_668402 67624239acyl-CoA:diacylglycerol acyltransferase 1-related enzyme[Cryptosporidium hominis TU502] AAP94208 33113253acyl-CoA:diacylglycerol acyltransferase 1-related enzyme [Toxoplasmagondii] AAP94209 33113255 acyl-CoA:diacylglycerol acyltransferase1-related enzyme [Toxoplasma gondii] XP_579557 62652535 PREDICTED:diacylglycerol O-acyltransferase 1 [Rattus norvegicus] BAC66171 29170489diacylglycerol acyltransferase [Mus musculus] Q9ERM3 18202872Diacylglycerol O-acyltransferase 1 (Diglyceride acyltransferase)AAL78366 18698659 acyl coenzyme A:diacylglycerol acyltransferase[Drosophila melanogaster] NP_995724 45552403 CG31991-PD, isoform D[Drosophila melanogaster] NP_724017 24584734 CG31991-PC, isoform C[Drosophila melanogaster] XP_858062 73974765 similar to DiacylglycerolO-acyltransferase 1 (ACAT related gene product 1) isoform 3 [Canisfamiliaris] XP_728984 82915156 hypothetical protein PY01256 [Plasmodiumyoelii yoelii str. 17XNL] CAG11944 47225461 unnamed protein product[Tetraodon nigroviridis] BAD27526 50199438 acyl-CoA:diacylglycerolacyltransferase [eukaryotic synthetic construct] XP_317656 31226099ENSANGP00000002281 [Anopheles gambiae str. PEST] AAV59457 55733950putative diacylglycerol acyltransferase [Oryza sativa (japonicacultivar-group)] EAL33593 54644853 GA16599-PA [Drosophila pseudoobscura]XP_678753 68073677 diacylglycerol O-acyltransferase [Plasmodium bergheistrain ANKA] XP_520014 55631434 PREDICTED: similar to DiacylglycerolO-acyltransferase 1 (Diglyceride acyltransferase) [Pan troglodytes]CAG10815 47219451 unnamed protein product [Tetraodon nigroviridis]XP_624754 66522700 PREDICTED: similar to ENSANGP00000002281 [Apismellifera] CAC69884 15620769 diacylglycerol acyltransferase I [Rattusnorvegicus] XP_686181 68363630 PREDICTED: similar to DiacylglycerolO-acyltransferase 1 (Diglyceride acyltransferase) [Danio rerio]XP_734008 70921323 diacylglycerol O-acyltransferase [Plasmodium chabaudichabaudi] XP_673128 68062248 hypothetical protein PB300300.00.0[Plasmodium berghei strain ANKA] AAS72376 45642963 acyl-CoA:cholesterolacyltransferase beta [Toxoplasma gondii] AAS72375 45642961acyl-CoA:cholesterol acyltransferase alpha [Toxoplasma gondii] NP_58614519074639 STEROL O-ACYLTRANSFERASE [Encephalitozoon cuniculi GB-M1]XP_640280 66812202 hypothetical protein DDB0205259 [Dictyosteliumdiscoideum] AAY40783 63376221 diacylglycerol acyltransferase [Brassicajuncea] XP_765774 71032265 diacylglycerol O-acyltransferase [Theileriaparva strain Muguga] Q876L2 34582301 Sterol O-acyltransferase 2(Sterol-ester synthase 2) XP_571260 58268208 sterol O-acyltransferase[Cryptococcus neoformans var. neoformans JEC21] EAL20032 50257323hypothetical protein CNBF3580 [Cryptococcus neoformans var. neoformansB-3501A] XP_954478 84999514 acyl transferase [Theileria annulata strainAnkara] XP_505086 50555355 hypothetical protein [Yarrowia lipolytica]NP_588558 19076058 hypothetical protein SPCP1E11.05c[Schizosaccharomyces pombe 972h-] AAC49441 1389739 acyl-CoA:sterolacyltransferase NP_014416 6324346 Acyl-CoA:sterol acyltransferase,isozyme of Are1p; Are2p [Saccharomyces cerevisiae] XP_750354 70991010sterol o-acyltransferase APE2 [Aspergillus fumigatus Af293] XP_38219246110268 hypothetical protein FG02016.1 [Gibberella zeae PH-1] BAE5493483764790 unnamed protein product [Aspergillus oryzae] XP_885914 76617939similar to Sterol O-acyltransferase 2 (Cholesterol acyltransferase 2)(ACAT-2) isoform 2 [Bos taurus] XP_591251 76617937 similar to SterolO-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) isoform 1[Bos taurus] BAC00846 21392392 AcylCoA:Cholesterol Acyltransferase 2[Rattus norvegicus] NP_649816 28571583 CG8112-PA [Drosophilamelanogaster] NP_666176 22122547 sterol O-acyltransferase 2 [Musmusculus] O88908 18202245 Sterol O-acyltransferase 2 (Cholesterolacyltransferase 2) (ACAT-2) XP_761502 71022545 hypothetical proteinUM05355.1 [Ustilago maydis 521] NP_714950 40254723 sterolO-acyltransferase 2 [Rattus norvegicus] EAQ86094 88178626 hypotheticalprotein CHGG_07347 [Chaetomium globosum CBS 148.51] XP_461395 50425599hypothetical protein DEHA0F25652g [Debaryomyces hansenii CBS767]XP_661812 67527926 hypothetical protein AN4208.2 [Aspergillus nidulansFGSC A4] AAH96091 64654094 Sterol O-acyltransferase 2 [Homo sapiens]O75908 18202149 Sterol O-acyltransferase 2 (Cholesterol acyltransferase2) (ACAT-2) AAH96090 64652990 Sterol O-acyltransferase 2 [Homo sapiens]AAK48829 13898623 acyl coenzyme A: cholesterol acyltransferase-2 [Homosapiens] XP_543637 73996435 PREDICTED: similar to sterolO-acyltransferase 2 [Canis familiaris] O77759 18202176 SterolO-acyltransferase 2 (Cholesterol acyltransferase 2) (ACAT-2) AAO3247428564191 ARE2 [Saccharomyces castellii] XP_323485 32405744 hypotheticalprotein [Neurospora crassa] NP_982606 45184888 AAR065Cp [Eremotheciumgossypii] NP_593708 19114620 hypothetical protein SPAC13G7.06[Schizosaccharomyces pombe 972h-] AAO32554 28564940 ARE2 [Saccharomyceskluyveri] EAL28962 54639560 GA20833-PA [Drosophila pseudoobscura]XP_449806 50294790 hypothetical protein CAGL0M10571g [Candida glabrataCBS138] NP_033256 84619697 sterol O-acyltransferase 1 [Mus musculus]Q61263 18202591 Sterol O-acyltransferase 1 (Cholesterolacyltransferase 1) (ACAT-1) BAC34925 26342537 unnamed protein product[Mus musculus] XP_452607 50305295 unnamed protein product [Kluyveromyceslactis] NP_001... 77735363 hypothetical protein LOC504287 [Bos taurus]Q60457 18202585 Sterol O-acyltransferase 1 (Cholesterolacyltransferase 1) (ACAT-1) XP_320321 58393811 ENSANGP00000016512[Anopheles gambiae str. PEST] XP_320320 58393809 ENSANGP00000016486[Anopheles gambiae str. PEST] O70536 18202126 Sterol O-acyltransferase 1(Cholesterol acyltransferase 1) (ACAT-1) XP_714776 68482533 acyl-CoAcholesterol acyltransferase [Candida albicans SC5314] P84285 56404462Sterol O-acyltransferase 2 (Sterol-ester synthase) (ASAT) AAH7791650416229 Soat1-prov protein [Xenopus laevis] XP_692855 68364838PREDICTED: similar to Soat1-prov protein [Danio rerio] CAI13574 55960156sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase)1 [Homo sapiens] AAL56227 18028942 cholesterol acyltransferase 1[Gorilla gorilla] AAL56228 18028944 cholesterol acyltransferase 1 [Pongopygmaeus] AAC37532 4878022 acyl-coenzyme A: cholesterol acyltransferase[Homo sapiens] 2201440A 1585676 acyl-CoA/cholesterol acyltransferaseQ876L3 34582302 Sterol O-acyltransferase 1 (Sterol-ester synthase 1)BAE01048 67969393 unnamed protein product [Macaca fascicularis]XP_514030 55588858 PREDICTED: hypothetical protein XP_514030 [Pantroglodytes] XP_547445 73961286 similar to Sterol O-acyltransferase 1(Cholesterol acyltransferase 1) (ACAT-1) [Canis familiaris] EAQ8461988177151 hypothetical protein CHGG_08633 [Chaetomium globosum CBS148.51] O77761 18202178 Sterol O-acyltransferase 1 (Cholesterolacyltransferase 1) (ACAT-1) XP_422267 50751122 PREDICTED: similar toSterol O-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1)[Gallus gallus] XP_693284 68392980 PREDICTED: similar to SterolO-acyltransferase 1 (Cholesterol acyltransferase 1) (ACAT-1) [Daniorerio] AAT92940 51013293 YCR048W [Saccharomyces cerevisiae] XP_95657685080625 hypothetical protein [Neurospora crassa N150] XP_62469166564061 PREDICTED: similar to ENSANGP00000016486 [Apis mellifera]CAF96514 47222847 unnamed protein product [Tetraodon nigroviridis]XP_788209 72085563 PREDICTED: similar to sterol O-acyltransferase 1[Strongylocentrotus purpuratus] XP_445307 50285757 unnamed proteinproduct [Candida glabrata] CAE70002 39596364 Hypothetical proteinCBG16409 [Caenorhabditis briggsae] CAG07990 47225647 unnamed proteinproduct [Tetraodon nigroviridis] NP_510623 17549960 B0395.2[Caenorhabditis elegans] AAX28331 76157393 SJCHGC04421 protein[Schistosoma japonicum] CAI96158 66347204 DiacylglycerolO-acyltransferase [Bubalus bubalis] XP_390039 46136695 hypotheticalprotein FG09863.1 [Gibberella zeae PH-1] XP_643169 66819019 hypotheticalprotein DDB0203882 [Dictyostelium discoideum] AAO53095 28850306hypothetical protein [Dictyostelium discoideum] AAB06959 1515472acyl-CoA:cholesterol acyltransferase [Oryctolagus cuniculus] NP_94561939933343 putative alginate o-acetyltransferase AlgI [Rhodopseudomonaspalustris CGA009] ZP_008... 77691302 Membrane bound O-acyl transferase,MBOAT [Rhodopseudomonas palustris BisB5] XP_465546 50908115 putative waxsynthase [Oryza sativa (japonica cultivar- group)]

TABLE 29 Examples of Prenyldiphosphate synthase polypeptides AccessionGI Description 29A: Bacteria Proteins that require a mitochondrialtargeting sequence ZP_009 . . . 83373595Trans-hexaprenyltranstransferase [Rhodobacter sphaeroides ATCC 17029]ZP_009 . . . 83371280 Trans-hexaprenyltranstransferase [Rhodobactersphaeroides ATCC 17025] CAD24417 20429105 decaprenyl diphosphatesynthase [Paracoccus zeaxanthinifaciens] ZP_010 . . . 85705714Geranylgeranyl pyrophosphate synthase/Polyprenyl synthetase [Roseovariussp. 217] ZP_010 . . . 84515724 decaprenyl diphosphate synthase[Loktanella vestfoldensis SKA53] YP_165582 56695234 decaprenyldiphosphate synthase [Silicibacter pomeroyi DSS-3] ZP_010 . . . 86139019decaprenyl diphosphate synthase [Roseobacter sp. MED193] ZP_009 . . .83941379 decaprenyl diphosphate synthase [Sulfitobacter sp. EE-36]ZP_009 . . . 83854856 decaprenyl diphosphate synthase [Sulfitobacter sp.NAS-14.1] ZP_006 . . . 69299873 Farnesyltranstransferase [Silicibactersp. TM1040] ZP_010 . . . 84683979 Geranylgeranyl pyrophosphatesynthase/Polyprenyl synthetase [Rhodobacterales bacterium HTCC2654]ZP_009 . . . 84500217 decaprenyl diphosphate synthase [Oceanicolabatsensis HTCC2597] ZP_009 . . . 83952381 decaprenyl diphosphatesynthase [Roseovarius nubinhibens ISM] ZP_006 . . . 69937106Trans-hexaprenyltranstransferase [Paracoccus denitrificans PD1222]ZP_005 . . . 68180845 Trans-hexaprenyltranstransferase [Jannaschia sp.CCS1] ZP_008 . . . 78495595 Polyprenyl synthetase [Rhodopseudomonaspalustris BisB18] AAY82368 67866738 decaprenyl diphosphate synthase[Agrobacterium tumefaciens] NP_353656 15887975 hypothetical proteinAGR_C_1125 [Agrobacterium tumefaciens str. C58] ZP_008 . . . 77688465Farnesyltranstransferase [Rhodopseudomonas palustris BisB5] NP_53133417934544 octaprenyl-diphosphate synthase [Agrobacterium tumefaciens str.C58] YP_484709 86748213 Farnesyltranstransferase [Rhodopseudomonaspalustris HaA2] AAP56240 37903500 decaprenyl diphosphate synthase[Agrobacterium tumefaciens] YP_192388 58040424 Decaprenyl diphosphatesynthase [Gluconobacter oxydans 621H] 29B: Subunit 1- Proteins thatcontain mitochondrial targeting sequence T43193 11279237trans-pentaprenyltranstransferase homolog - fission yeast(Schizosaccharomyces pombe) AAD28559 4732024 trans-prenyltransferase[Homo sapiens] AAI07275 78070698 Trans-prenyltransferase [Mus musculus]BAE48216 81157931 subunit 1 of decaprenyl diphosphate synthase [Homosapiens] AAH49211 29165656 PDSS1 protein [Homo sapiens] Q33DR2 85700953Decaprenyl-diphosphate synthase subunit 1 (Solanesyl-diphosphatesynthase subunit 1) (Trans-prenyltransferase) XP_507706 55633583PREDICTED: similar to TPRT protein [Pan troglodytes] XP_586717 76632198PREDICTED: similar to trans-prenyltransferase [Bos taurus] XP_84990873948851 PREDICTED: similar to trans-prenyltransferase [Canisfamiliaris] 29C: Subunit 2- Proteins that contain mitochondrialtargeting sequence O13851 60389474 Decaprenyl-diphosphate synthasesubunit 2 (Decaprenyl pyrophosphate synthetase subunit 2) BAE4821881157935 subunit 2 of solanesyl diphosphate synthase [Mus musculus]BAE48217 81157933 subunit 2 of decaprenyl diphosphate synthase [Homosapiens]

TABLE 30 Examples of PHB-Polyprenyltransferase polypeptides GI PROTEINDESCRIPTION 51013645 YNR041C [Saccharomyces cerevisiae] 50285815 unnamedprotein product [Candida glabrata] 50311051 unnamed protein product[Kluyveromyces lactis] 45200866 AGL231Wp [Eremothecium gossypii]50555263 hypothetical protein [Yarrowia lipolytica] 68473193para-hydroxybenzoate: polyprenyl transferase [Candida albicans SC5314]50410039 hypothetical protein DEHA0A14212g [Debaryomyces hanseniiCBS767] 83769349 unnamed protein product [Aspergillus oryzae] 70994900para-hydroxybenzoate-polyprenyltransferase precursor [Aspergillusfumigatus Af293] 19114131 hypothetical protein SPAC56F8.04c[Schizosaccharomyces pombe 972h-] 39973573 hypothetical proteinMG01067.4 [Magnaporthe grisea 70-15] 85078920 protein related topara-hydroxybenzoate polyprenyltransferase precursor [Neurospora crassaN150] 76660839 PREDICTED: similar topara-hydroxybenzoate-polyprenyltransferase, mitochondrial [Bos taurus]52138578 para-hydroxybenzoate-polyprenyltransferase, mitochondrial [Homosapiens] 18088424 COQ2 protein [Homo sapiens] 47221448 unnamed proteinproduct [Tetraodon nigroviridis] 58385249 ENSANGP00000012220 [Anophelesgambiae str. PEST] 50746583 PREDICTED: similar to hypothetical proteinCL640 [Gallus gallus] 54638587 GA21912-PA [Drosophila pseudoobscura]21355567 CG9613-PA [Drosophila melanogaster] 71005862 hypotheticalprotein UM01450.1 [Ustilago maydis 521]

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1-57. (canceled)
 58. A method of producing a carotenoid, the methodcomprising cultivating a genetically engineered fungus under conditionsthat allow production of the carotenoid; wherein the fungus ischaracterized by: i. the fungus is oleaginous in that it can accumulatelipid to at least about 20% of its dry cell weight; and ii. the fungusproduces at least one carotenoid, and can accumulate the producedcarotenoid to at least about 1% of its dry cell weight; wherein thefungus comprises at least one modification selected from the groupconsisting of carotenogenic modifications, oleaginic modifications, andcombinations thereof, and wherein the at least one modification altersoleaginicity of the fungus, confers to the fungus oleaginy, confers tothe fungus the ability to produce the at least one carotenoid to a levelat least about 1% of its dry cell weight, or confers to the fungus theability to produce at least one carotenoid which the fungus does notnaturally produce. 59-63. (canceled)
 64. A method of producing acarotenoid, the method comprising cultivating a genetically engineeredYarrowia fungus under conditions that allow production of thecarotenoid; wherein the genetically engineered Yarrowia fungus ischaracterized by: i. the fungus is oleaginous in that it can accumulatelipid to at least 20% of its dry cell weight; and ii. as a result of thegenetic engineering, the Yarrowia fungus produces at least onecarotenoid, and can accumulate the produced carotenoid to at least 1% ofits dry cell weight.
 65. The method of claim 58, wherein the methodcomprises a second step of isolating the produced carotenoid.
 66. Themethod of claim 65 wherein the step of isolating comprises fractionatingthe cultivation medium to obtain at least one carotenoid-enrichedfraction.
 67. The method of claim 58 wherein: the step of cultivatingcomprises cultivating the fungus under conditions that allowaccumulation of the carotenoid in cytoplasmic oil bodies; and the stepof isolating comprises isolating oil derived from the cytoplasmic oilbodies.
 68. The method of claim 58 wherein the carotenoid is selectedfrom the group consisting of astaxanthin, β-carotene, canthaxanthin,zeaxanthin, lutein, lycopene, and combinations thereof.
 69. The methodof claim 58 wherein the carotenoid comprises astaxanthin.
 70. The methodof claim 58 wherein the carotenoid comprises β-carotene.
 71. The methodof claim 58 wherein the carotenoid comprises canthaxanthin.
 72. Themethod of claim 58 wherein the carotenoid comprises zeaxanthin.
 73. Themethod of claim 64, wherein the method comprises a second step ofisolating the produced carotenoid.
 74. The method of claim 73 whereinthe step of isolating comprises fractionating the cultivation medium toobtain at least one carotenoid-enriched fraction.
 75. The method ofclaim 64 wherein: the step of cultivating comprises cultivating thefungus under conditions that allow accumulation of the carotenoid incytoplasmic oil bodies; and the step of isolating comprises isolatingoil derived from the cytoplasmic oil bodies.
 76. The method of claim 64wherein the carotenoid is selected from the group consisting ofastaxanthin, β-carotene, canthaxanthin, zeaxanthin, lutein, lycopene,and combinations thereof.
 77. The method of claim 64 wherein thecarotenoid comprises astaxanthin.
 78. The method of claim 64 wherein thecarotenoid comprises β-carotene.
 79. The method of claim 64 wherein thecarotenoid comprises canthaxanthin.
 80. The method of claim 64 whereinthe carotenoid comprises zeaxanthin.
 81. The carotenoid produced by themethod of claim 58.